Changes of AsA and GSH in BvM14 leaves under salt stress
Recent studies showed that AsA and GSH are major antioxidants in plant salt stress response (Navrot et al. 2011;Lin et al. 2020;Khan et al. 2020), here we measured changes of two major antioxidants AsA and GSH at 0, 5, 10, 20, 30, 60 and 90 min after 0, 200, 400 mM NaCl treatments. As shown in Figure 1, under control conditions, the contents of AsA and GSH in BvM14 leaves maintained at fairly constant levels during the 90 min of assay time. Compared to control conditions, both the AsA and GSH contents reached maximum after 30 min of 200 mM NaCl stress. While after 60 min of 400 mM NaCl stress, both the AsA and GSH contents reached the peak level (Fig.1). The results clearly showed that salt stress caused significant cellular redox changes as early as 10 min after the treatment. Based on the AsA and GSH changes, we selected samples collected at 30 min and 60 min of 200 mM and 400 mM NaCl conditions, respectively, for iodoTMTRAQ-based redox proteomics.
Identification of differential proteins and different redox proteins in response to salt stress
Using iodoTMTRAQ LC-MS/MS and database searching, a total of 1290 proteins were identified in BvM14 leaves (Supplemental Table S1). Eighty proteins were differentially changed in abundance (based on iTRAQ reporter fold change>1.2, or <0.8, p<0.05) in salt-treated samples compared to the control samples (Supplemental Table S2). Only four differential proteins were identified under the 200 mM NaCl treatment, while 77 were identified under the 400 mM NaCl treatment. Functional classification of the differential proteins revealed the following distribution: metabolism (6.3%), protein synthesis (27.4%), transport (6.3%), stress and defense (2.5%), ROS homeostasis (7.5%), protein stability and turnover (5%), photosynthesis (5%), transcription related (6.3%) and unknown (33.7%) (Fig. 2A). The subcellular locations of the 80 differential proteins were classified to the chloroplast (32.7%), cytoplasm (11.5%), cytosol (1.9%), mitochondrial (7.7%), nuclear (42.4%), plasma membrane (1.9%) and vacuole (1.9%) (Fig. 2B).
Based on iodoTMT reporter intensities, we identified 42 proteins with significant redox changes in response to salt stress (Supplemental Table S3). Here are the functional categories of the differential redox proteins: metabolism (9.5%), transport (16.7%), biosynthesis (19.1%), transcription related (2.4%), signal transduction (4.8%), stress and defense (2.4%), ROS homeostasis (7.1%), photosynthesis (26.2%) and unknown (11.8%) (Fig. 2C). The subcellular localizations of the redox proteins were classified to the chloroplast (55%), cytoplasm (2.5%), cytoskeleton (2.5%), mitochondrial (2.5%), nuclear (7.5%), plasma membrane (5%), extracellular (22.5%) and vacuole (2.5%) (Fig. 2D). Among the 42 differential redox proteins, four were identified under 200 mM NaCl treatment, and 40 were identified under 400 mM NaCl treatment. There were 31 oxidized and 18 reduced cysteine residues in the redox proteins (Table 1).
Mapping redox responsive cysteine residues in the BvM14 response to salt stress
With the acquired MS/MS spectra, a total of 49 redox responsive peptides were identified in the 42 redox proteins (Supplemental Tables S3). In these peptides, the redox modified cysteine residues could be mapped. In Figure 3, the MS/MS spectra of two redox peptides derived from ATP synthase (731341013) and malate dehydrogenase (731329081) were shown as examples (Fig. 3A, B).
Transcriptional analysis of differential redox proteins and differential proteins
To test how transcriptional level changes correlate with protein level and redox protein level, 11 differential proteins and seven differential redox protein were selected for analysis of their gene transcriptional level changes. The Real-time PCR primer sequences can be found in Supplemental Table S4. We categoriezed the transcriptional expression patterns of these genes into six groups based on their functions (Fig. 4, Supplemental Table 4,). The first group proteins were involved in photosynthesis, including Rubisco LSU, Fd, Fd-1. The second group proteins were involved in ROS homeostasis, including Clot, Cys, PDIL1-1, CBSX3, EGC1, peroxidase (POD), Trx3-1, TrxH1, and TL29. The third group belonged to transport-related pathway including nsLTP, atpC protein. The fourth group DLD1 proteins belonged to metabolism. The fifth group RNase LE proteins belonged to biosynthesis. The last group proteins were stress and defense cascade, including DDR48 and DUF642.
Among the 18 genes encoding for the differential proteins, the transcriptional levels of 12 genes were coincide with the corresponding redox level trends and total protein level trends (Supplemental Table S5). The transcriptional levels of ATP synthase epsilon chain (atpC), ferredoxin (Fd-1), POD and thioredoxin-like 3-1 (Trx3-1) showed different trends with the corresponding redox changes, while the extracellular ribonuclease LE-like (RNase LE), DUF642 and EG45-like domain containing protein 1(EGC1) showed the same trend at both the protein level and transcriptional level (Supplemental Table S5).
A review of potential salt stress response mechanisms in BvM14
On the basis of the aforementioned results, we proposed a potential mechanism in the s BvM14 response to short-term salt stress (Fig. 5, Supplemental Table S6). The differential redox proteins and total proteins put into context of subcellular locations and pathways under salt stress. The key pathways in Figure 5 include ROS homeostasis, photosynthesis, stress and defense, transport related processes. Nevertheless, our results highlight the following potential mechanisms under salt stress: Salt stress leads to ROS production and oxidative stress, which lead to redox changes in microenvironment of cytoplasm and various organelles, resulting in redox PTMs of proteins in biochemical pathways dominated by photosynthesis and ROS homeostasis. The redox PTMs revealed in this study may play important regulatory roles in the BvM14 salt stress response and contribute to the development of salt stress tolerance.