Nitrogen (N) is one of the basic nutrients and signals for plant development and deficiency of it would always limit the productions of crops in the field. Quantitative research on expression of N-stress responsive proteins on a proteome level remains elusive. In order to gain a deep insight into the proteins responding to nitrogen stress in rapeseed (
Nitrogen (N) is one of the essential nutrients for plants serving as a constituent of nucleotides and proteins [
Many studies indicate that plants have evolved complicated strategies to adapt themselves to either short-term or long-term N-deficiency stress [
Comparative proteomic analysis between untreated and stress-treated conditions has been used to identify major proteins involved in stress responses in rice [
Seeds of rapeseed were surface-sterilized for 20 min in 0.5% (w/v) sodium hypochlorite solution and washed at least three times by distilled water. After that, seeds were germinated on moistened gauze in a black plastic tray filled with deionized water. Rapeseeds were cultivated as described by Basu et al. [
After the emergence of the first true leaf, seedlings were transferred into a modified system consisting of four different N concentrations respectively: 0%, 10%, 50% and 100% N element relative content compared with standard MS media (100% N element relative content), where N element was supplied in the form of ammonium nitrate (NH4NO3), for 18 days. The MS solution [
After N treatments, the leaf, stem and root were sampled respectively for protein extraction, according to the description by Hurkman et al. [
Protein solution from each sample was treated successively with 5 mmol/L dithiothreitol at 56°C for 30 min and alkylated with 11 mmol/L iodoacetamide at RT in darkness for 15 min. The protein sample was then diluted by 100 mmol/L triethyl ammonium bicarbonate (TEAB) to make sure urea concentration ≤2 mol/L. Digestion was performed by adding trypsin at a 1:50 (trypsin:protein) ratio at 37°C overnight; then at a 1:100 (trypsin:protein) ratio for another 4 h.
After trypsin digestion, peptide was desalted by Strata X C18 SPE column (Phenomenex, California, USA) and vacuum-dried. Peptide was reconstituted in 0.5 M TEAB and labeled according to the instruction for TMT kit (ThermoFisher, Massachusetts, USA).
The tryptic peptides were fractionated into fractions by high pH reverse-phase HPLC using Agilent 300 Extend C18 column as the earlier report [
The generated data was then processed using Maxquant search engine (v.1.5.2.8), with the same parameters to Sun et al. [
Proteins with at least a two-fold change in expression level between treated sample and control and a
To investigate the effect of N-deficiency stress on rapeseed leaves, the contents of chlorophyll (Chl) and malonyldialdehyde (MDA), and the enzyme activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) were determined. Leaves under NL0.1, NL0.5 and NL1 treatments were sampled, respectively. 0.08 g leaves from each sample were collected and then the Chl content was determined [
To gain a global view of the N-stress responsive proteome in rapeseed, seedlings were treated under four N concentration conditions. Samples of root, stem and leaf were separately named as NR, NS and NL, respectively; while 0%, 10%, 50%, 100% of relative N concentration treatments were assigned as N0, N0.1, N0.5 and N1, respectively. In N-free condition (NS0), stem failed to be sampled because they were too puny to be collected.
In all rapeseed samples, the relative concentration of N at 50% was the appropriate concentration for rapeseed seedlings in our test, whereas either higher or lower N concentration conditions would limit the development of rapeseed at the two-leaf or five-leaf stage in N0, N0.1 and N1 condition (
Compared with the control (N0.5), samples in NR0, NR0.1 and NR1 obtained 146, 109 and 64 proteins increasing their expression abundance, respectively; while 177, 130 and 101 proteins were down-regulated in the three treatments, respectively (
To deeply understand the functions of the identified DAPs in N-stress in roots, we further performed the Gene Ontology (GO) analysis. Functional classifications were defined using level 2 GO terms. Finally, all of the proteins were categorized into various groups according to their GO terms. In root, most of those DAPs were involved in metabolism processes, cellular processes and single-organism processes under different N-stress conditions. Surprisingly, over 60 proteins related to metabolism were affected in other three N concentration conditions when compared with NR0.5 as control (
Due to the fundamental role of N element, plants could adjust the basic metabolism processes in response to different N concentrations. In order to gain an insight into the cellular processes affected by N-stress, functional enrichment analysis was performed to identify GO terms, including biological process (BP), molecular function (MF) and cellular component (CC) ontologies. GO enrichment analysis of DAPs between NR0 and NR0.5 suggested that the photosynthesis process, response to stimulus, especially for oxidative stress, lipid metabolic process as well as carbohydrate metabolic process were all affected by N-deficiency condition (
DAPs identified from leaf tissue were also classified based on their GO analysis (
In stem tissue, only samples cultivated in MS medium with 10%, 50% and 100% of N concentrations, i.e., NS0.1, NS0.5 and NS1, were collected because of the failure of stem tissue collection from the rapeseed seedling in N-free (NS0) samples. Comparing with control (NS0.5), less than 100 DAPs were obtained in the treatment of N-deficiency (
As described above, a large majority of DAPs were identified based on different tissues and various N concentration treatments (
Up/down | Tissue | N deficiency | N excess |
---|---|---|---|
Up-regulated | Root | A0A078H2C2, A0A078H752, A0A078J6D0, A0A078J902, A0A078E3T7 | A0A078DDZ0, A0A078F768, A0A078EF82, A0A078ISD8, A0A078JBG2, A0A078EAK5, A0A078GSP3, A0A078D2R9, A0A078FA32, A0A078HV12, A0A078FU54, A0A078BWB3, A0A078CLG8, A0A078D3E6, A0A078GX94, A0A078H4N9, A0A078DI76, A0A078JLV5, A0A078HXQ3, A0A078ITD6, A0A078CVP2, A0A078EGZ0, A0A078ES97, A0A078J7M7, A0A078HVP0, A0A078GQ84, A0A078HJF4, A0A078J785, A0A078I0V1, A0A078JHY0, A0A078HU21, A0A078CTQ1, A0A078D0B4, A0A078HCV6, A0A078CR59, A0A078E3F9, A0A078IDV5, A0A078J902, A0A078EKV7, A0A078HXV1, A0A078I263, A0A078GB75, A0A078GSS0, A0A078F4Q8, A0A078DUN5, A0A078HZV6, A0A078F6S6, A0A078D4M6, A0A078JZM9, A0A078HBR9, A0A078IKT4, A0A078GC16, A0A078IG72, A0A078ERH8, A0A078DP99, A0A078EAG3, A0A078GQ95, A0A078H752, A0A078JFR2, A0A078IGH2, A0A078H1R4, A0A078GMU3, A0A078H2C2, A0A078CAI7 |
Stem | A0A078JW50, A0A078FCM1, A0A078J4Z5, A0A078FNY0, A0A078JJB9, A0A078CUP3, A0A078DAQ8, A0A078H2C2, A0A078GCN9, A0A078IQQ0, A0A078CLB6, A0A078EW55, A0A078CBH6, A0A078IKT4, A0A078H752, A0A078GU87, A0A078FB75, A0A078CW34, A0A078J624, A0A078ISZ0, A0A078HN33, A0A078JAV8, A0A078J0B7, A0A078IGP0, A0A078J6D0, A0A078F0B8, A0A078IQH8, A0A078CU21, A0A078J902, A0A078E3T7, A0A078JZB1, A0A078BU25, A0A078JTE2, A0A078D831, A0A078IFF7, A0A078CMW4, A0A078FKB8, A0A078HE05, A0A078ES15, A0A078G0C3, A0A078JCX4, A0A078HRM3, A0A078IZ52, A0A078HCQ8 | A0A078EIS2, A0A078D4S4, A0A078JW15, A0A078FLD9, A0A078IQE7, A0A078CWV1, A0A078H752, A0A078E1P2, A0A078EWE0, T1SRR1, A0A078H6W1, A0A078J344, A0A078D0Z9, A0A078FX05, A0A078JFH5, A0A078E586, A0A078F4Q8, A0A078GX90, A0A078J9V8, A0A078FQD0, A0A078JZM9, A0A078DD40, A0A078H7D9, A0A078J902, A0A078I784, A0A078GU91, A0A078FNY0, A0A078EW55, A0A078HN30, A0A078H5H9, A0A078D829, A0A078JFZ1, A0A078GC16, A0A078CKB1, A0A078H3S0, A0A078IKT4, A0A078F3P0, A0A078CW34, A0A078D0B4, A0A078DYG3, A0A078H8Q0, A0A078H2C2, A0A078HLY0, A0A078CAI7 | |
Leaf | A0A078CBH6, A0A078JTE2, A0A078FI39, A0A078HI09, A0A078DAQ8, A0A078JZB1, A0A078I9R1, A0A078JHG6, A0A078J0B7, A0A078H7D9, A0A078I083, A0A078FNY0, A0A078FTP1, A0A078F776, A0A078G2S9, A0A078CYM4 | A0A078HM16, A0A078JHG6, A0A078G4Z5, A0A078JFX9, A0A078E4M0, A0A078E0T0, A0A078DD79, A0A078D0Z9, A0A078J1Z0, A0A078D6Q8, A0A078DI76, A0A078DM76, A0A078FCW6, A0A078HDH3, A0A078ISZ0, A0A078E9D0, A0A078FX05, A0A078HAT9, A0A078F5M5, A0A078HCG2, A0A078IJ41, A0A078G3K2, A0A078DVL6, A0A078H8V8, A0A078DXZ1, A0A078J9J8, A0A078GTX9, A0A078IJK1, A0A078EIS2, A0A078HLJ2, A0A078CKB1, A0A078HW44, A0A078I9R1, A0A078EAG3, A0A078HN15, A0A078IG72, A0A078IR26, A0A078GX74, A0A078HV35, A0A078CES8, A0A078C9K6, A0A078GNJ7, A0A078CD81, A0A078C3H6, A0A078FA43, A0A078I8C8, A0A078DYG3, A0A078H4Z3, A0A078FQD0, A0A078DWY2, A0A078CXA7, A0A078H7D9, A0A078C7Q5, A0A078E586, A0A078HLY0, A0A078IQH8, A0A078CAI7 | |
Root | A0A078HH74, A0A078GLF6, A0A078K038, A0A078E4C7, A0A078HT85, A0A078HFR0, A0A078J7K4, A0A078FRF3, A0A078G2P1, A0A078ES77 | A0A078CXA7, A0A078FUK6, A0A078I8C8, A0A078F1A3, F8SPG0, A0A078GQA6, A0A078BXN2, A0A078C3H6, A0A078CX90, A0A078HTA8, A0A078DQ41, A0A078BXA8, A0A078INR3, A0A078E9I5, A0A078FA61, A0A078HV35, A0A078EHL4, A0A078GP82, A0A078I427, A0A078IC76, A0A078HT85, A0A078JPM1, A0A078JEK8, A0A078G355, A0A078BUC2, A0A078JPL2, A0A078G3T1, A0A078CIE5, A0A078CQS6, A0A078CU21, A0A078EIW9, A0A078GWX8, D1L8Q5, A0A078F0S8, A0A078EYI7, A0A078E382, A0A078FK57, A0A078HRZ2, A0A078E3M9, A0A078GUT8, A0A078C9H1, A0A078D5F5, A0A078EL48, A0A078H0R6, A0A078D197, A0A078FV38, A0A078F4A0, A0A078HBB1, A0A078JRE8, A0A078HKY6, A0A078DAM0, A0A078C7W5, A0A078JBN4, A0A078BVH9, A0A078FA16, A0A078C335, A0A078FRY4, A0A078J564, A0A078DDH5, A0A078I0B6, A0A078DJH8, A0A078JMK0, A0A078D660, A0A078BTL6, A0A078CY15, A0A078J3S0, A0A078HDS3, A0A078FC13, A0A078JW15, A0A078CC83, A0A078IIX1, A0A078IX79, A0A078E317, A0A078CNX8, A0A078GAR9, A0A078EWE0, A0A078DY92, A0A078EB95, A0A078I2K9, A0A078GXV7, A0A078II13, A0A078I433, A0A078IHT2, A0A078I8C4, I7DDY9, A0A078DV43, A0A078GX98, A0A078HK20, A0A078GGI1, A0A078JFC8, A0A078HTY5, A0A078DZ35, A0A078BZY8, A0A078DV07, A0A078DB47, A0A078GNY7, A0A078HFQ8, A0A078JXS4, A0A078GEW2, A0A078HSN4, A0A078IZK1 | |
Down-regulated | Stem | A0A078CEA8, Q42386, A0A078D1N3, A0A078HV35, A0A078JP31, A0A078FUI0, A0A078HMH2, A0A078EU97, A0A078HH74, A0A078CAK0, A0A078D821, A0A078CIV5, A0A078GFI9, A0A078GBK6, A0A078GLF6, A0A078JRR6, A0A078FRA0, A0A078DEW9, A0A078K038, A0A078GDJ9, A0A078H2Z4, A0A078F572, A0A078C9K6, A0A078IWY9, A0A078JD19, A0A078E4C7, A0A078H1B6, A0A078EWP0, A0A078HYD7, A0A078CPB3, A0A078C7M5, A0A078HT85, A0A078HFR0, A0A078J7K4, A0A078FAM4, A0A078HLA3, A0A078JQ95, A0A078EWN3, A0A078EDE0, A0A078F710, A0A078CJN2, A0A078FRF3, A0A078DPS3, A0A078CT08, A0A078E9W6, Q43392, A0A078JQH4, A0A078J0L4, A0A078G2P1, A0A078ES77 | G1EIM2, A0A078C3H6, F8SPG0, A0A078HH28, A0A078HK20, A0A078EJG4, A0A078JEK8, A0A078HTY5, A0A078HT85, A0A078C9K6, A0A078CF19, A0A078E5Y0, A0A078G963, A0A078I8C4, A0A078CQS8, A0A078HMK1, A0A078C7W5, A0A078HV35, A0A078IJS3, A0A078C6A5, A0A078I8C8, A0A078I250, A0A078IA81, A0A078IJ50, A0A078CRC2, A0A078DVL1, A0A078DY92, A0A078HRZ2, A0A078DV43, A0A078C7M5, A0A078I3H1, A0A078D803, A0A078JDL6, A0A078EL76 |
Leaf | A0A078E8P8, A0A078BW01, A0A078FMH7, A0A078CT08, A0A078CPB3, A0A078ICU9, A0A078IWH9, A0A078E6U0, A0A078F8I0, A0A078I5B6, A0A078G2P1, A0A078C1D6, A0A078GUK1, A0A078J7K4, A0A078ES77, A0A078D1N3, A0A078GU87, A0A078HID8, A0A078FBV1, A0A078GQS2 | A0A078IJS3, A0A078C9M5, A0A078E5Y0, A0A078FK32, A0A078BTL6, A0A078G280, A0A078HC25, A0A078DC38, A0A078F4V4, A0A078IBU1, A0A078GSP3, A0A078C5V6, A0A078EL37, A0A078GT29, A0A078EYX7, A0A078JIG3, A0A078E3M2, A0A078HV12, A0A078E8Q8, A0A078HTY5, A0A078FAM4, A0A078F2Y3, A0A078CF19, A0A078C1D6, A0A078H2C2, A0A078IA81, A0A078CND1, A0A078I5B6, A0A078C6A5, A0A078GBX7, A0A078IG50, A0A078JNF7, A0A078I8C4, A0A078FLD9, Q42625, A0A078IC76, A0A078GKB0, A0A078CU52, A0A078C253, A0A078IR76, A0A078J7K4, A0A078GRY2, A0A078FEK1, A0A078HWP0, A0A078EDR4, A0A078J1J0, A0A078J257, A0A078F366, A0A078GGZ1, A0A078IWI1, A0A078F9V9, A0A078EWE0 |
Note: N-deficiency stood for N0 and N0.1. The DAPs in this category were those which expressed differentially to N0.5 in both N0 and N0.1. For the stem, only DAPs under N0.1 treatment in stem (NS0.1) were identified, because the samples were not collected. N-excess treatment stood for N1 treatment.
Based on the results obtained above, the photosynthesis process, oxidative stress and hydrogen peroxide metabolic process of rapeseed were significantly affected by the N-stress. To further ascertain these changes resulting from the different N concentrations, the contents of protein in leaves, chlorophylls (chl a, chl b and total chl), CAT, SOD, POD and MDA were analyzed. Our results indicated that both N0.1 and N1 could restrain the protein biosynthesis when compared with the control (N0.5). The chlorophyll (chlorophyll a, chlorophyll b and total chlorophyll) contents analyses showed that they were increased with the increase of N concentrations, and these suggested that the N content positively regulated the synthesis of chlorophylls (
Treatment | Chl a (mg/g) ± SD | Chl b (mg/g) ± SD | Total Chl (mg/g) ± SD |
---|---|---|---|
NL0.1 | 0.910c ± 0.023 | 0.339a ± 0.002 | 1.249b ± 0.006 |
NL0.5 | 0.974a ± 0.012 | 0.356a ± 0.008 | 1.330a ± 0.020 |
NL1 | 1.023b ± 0.004 | 0.409b ± 0.002 | 1.432c ± 0.006 |
Treatment | CAT (U/mg prot) |
POD (U/mg prot) |
SOD (U/mg prot) |
MDA (nmol/mg prot) |
---|---|---|---|---|
NL0.1 | 10.120b ± 0.726 | 57.803b ± 6.886 | 152.296b ± 1.594 | 3.900b ± 0.502 |
NL0.5 | 3.050a ± 0.191 | 17.683a ± 1.363 | 86.313a ± 1.481 | 2.731a ± 0.333 |
NL1 | 3.849a ± 0.731 | 16.586a ± 1.378 | 139.223b ± 6.159 | 2.499a ± 0.322 |
Nitrogen is one of the essential nutrients for life and in plants. Much of the total nitrogen is used in chlorophyll molecules, which are essential for photosynthesis and proper development. The N-starved
Previous results have proved that over-accumulation of reducing equivalents and elevated lipid degradation induced the excess ROS molecules under N-deficient conditions [
Rapeseed production has undergone a rapid increase in recent decades due to the abundant application of N fertilizer. However, on the other hand, the application of N fertilizer burdened farmers and led to well-documented environment problems [
Nitrogen is an essential nutrient to plant growth and development. Because of the complex reasons contributing to the loss of N in soil, plants must cope with the variation of N concentrations in soil and other environment to acquire proper N for themselves. N deficiency has been proved to negatively regulate growth or development [
Proteomic changes induced by N-stress are shown in their expression profiles in rapeseed tissues under several N-stresses conditions. Taking 50% N concentration as control, a total of 239 and 165 proteins significantly changed their expression levels in the NR0.1 and NR1 samples compared to NR0.5. There were only 124 and 74 DAPs separately identified in NS0.1 and NS0.5 samples, and 192 and 109 proteins were relatively affected by N-deficient stress in NL0.1 and NL0.5 samples. Functional classification and GO enrichment analysis illustrated that proteins related to metabolism and cellular processes accounted for two main portions of all DAPs regardless of tissue resource, including many key basic metabolic processes such as amino acid, protein and carbohydrate metabolic processes.
Eight proteins markedly changed their expression in high-N stress in all three tissues. Among them, ALIS1 (A0A078CAI7), the protein that catalyzes flipping of phospholipids across cellular membranes and contributes to vesicle biogenesis in the secretory and endocytic pathways [
In N-deficiency stress, the expressions of 14 proteins were universally affected regardless of tissue source: A0A078CMW4, A0A078CT08, A0A078D1N3, A0A078EWN3, A0A078EWP0, A0A078F710, A0A078G2P1, A0A078GLF6, A0A078H2C2, A0A078HV35, A0A078IQH8, A0A078J7K4, A0A078FCM1 and A0A078J4Z5. Among them, two proteins also changed their expression in response to high-N stress: A0A078H2C2 and A0A078HV35. While N-deficiency stress repressed the expression of A0A078H2C2 in root and stem, high-N stress induced more of it than the control. A0A078H2C2 is one protease enzyme involved in macromolecule and organic substance metabolic processes, and A0A078HV35 is a transcription factor that plays an important role in plants development. Low-N stress enhanced the expression of TF25, which contributes to cell death, in root and stem, while high-N reduced its expression. A0A078H2C2 is a protease enzyme involved in protein metabolic processes with metalloendopeptidase activity. A0A078HV35 is a transcription factor that plays an important role in plant development, which specifically increased its abundance in leaf tissues. Those results showed the common and specific response to N-stress in the three different tissues whereas N-deficient and high-N stress all affect the primer metabolic process in rapeseed.
The photosynthesis capacity of leaves has been reported related to the nitrogen content of plants, mainly because of the requirement of nitrogen in leaf proteins that are involved in the Calvin cycle [
Comparative proteomic analysis also indicated some proteins related to the photosynthesis process that were affected by the N concentration in the environment. N-deficient rapeseed had substantially changed the abundance of proteins that are involved in the photosynthetic light reaction, electron transport chain, carbon fixation, and other related regulation processes. Rapeseed in high-N stress conditions significantly decreased the abundance level of proteins such as photosystem I subunit VII, antenna protein, photosynthetic membrane protein and psbQ. Further analysis between N-free and control (NL0.5) samples demonstrated that the abundance of proteins required for the photosynthesis process was greatly up-regulated compared with N-deficient condition.
A large set of proteins related to biotic and abiotic stress-response processes were identified under N stress. Further GO enrichment analysis of DAPs in N-deficient condition indicated that six DAPs in N-deficient condition also responded to abiotic and biotic stress.
In N-deficient condition (NL0.1), more proteins were affected by N-starvation stress. A0A078BWD3, dehydrin ERD14-like protein, was specifically down-regulated in rapeseed root in N-deficient stress condition, and much more reduction was detected in N-free condition. A0A078J0B7 is one of the CHB4 family protein that is involved in defense against chitin-containing fungal pathogens, and it was only significantly up-regulated in rapeseed stem tissue. Besides those proteins induced by N-deficient stress, we also found some stress-related proteins that were down-regulated, such as MLP-like protein [
N-stress is well-documented to cause plant oxidative stress. In this study, many peroxidase proteins involved in hydrogen peroxide catabolic process were affected by N-deficient stress and high-N stress, such as A0A078IQH8, A0A078CES8, A0A078G4Z5, A0A078DYG3, A0A078HM16 and A0A078C7Q5. Peroxidase protein [
Further analysis showed that MDA content increased in N-deficient conditions and decreased in N-high conditions. Plants have evolved many strategies to defend themselves from oxidative stress with several antioxidant enzymes, such as POD, SOD and CAT. Our results showed the enhanced enzyme activity of those antioxidant proteins under N-stress, especially in N-deficient conditions. The activity of several antioxidant enzymes, including CAT, SOD and POD, was measured in those leaf samples. In addition, the content of MDA and the activities of antioxidant enzymes under N-deficient conditions were gradually increased, suggesting the aggravation of oxidative stress with the proceeding N-deficiency stress.
This study identified the N-stress responsive genes in rapeseed by investigation of proteomics after various N-concentration treatments. A large set of genes significantly changed their protein abundance under low- and high-N stress conditions, including proteins involved in basic metabolic processes and stress-response processes, which are essential for plant development. Further analysis revealed that genes/proteins associated with carbohydrate metabolism, nucleotide metabolism, amino acid metabolism, disease defense, oxidative stress response and photosynthesis were involved in N-stress responses. This study shed light on further research for understanding the N-stress response mechanisms in