Protein Changes in Response to Lead Stress of Lead-Tolerant and Lead-Sensitive Industrial Hemp Using SWATH Technology
1School of Agriculture, Yunnan University, Kunming, Yunnan 650504, China; chenxia3362623@163.com (C.X.); lhyndx@hotmail.com (L.H.); wsyjy198797@163.com (Y.Y.); denggang1986@ynu.edu.cn (D.G.)
2College of Agriculture and Life Sciences, Kunming University, Kunming, Yunnan 650241, China; chenxia3362623@163.com
3Industrial Crop Research Institute, Yunnan Academy of Agricultural Sciences, Kunming, Yunnan 650205, China; jingzuosuo@163.com
4Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan 571101, China; hxalong@gmail.com
*Correspondence: denggang1986@ynu.edu.cn; Tel.: +86-871-65031539Abstract
Hemp is a Pb-tolerant and Pb-accumulating plant and the study of its tolerance mechanisms could facilitate the breeding of hemp with enhanced Pb tolerance and accumulation. In the present study, we took advantage of sequential window acquisition of all theoretical mass spectra (SWATH) technology to study the difference in proteomics between the leaves of Pb-tolerant seed-type hemp variety Bamahuoma (BM) and the Pb-sensitive fiber-type hemp variety Yunma 1 (Y1) under Pb stress (3 g/kg soil). A total of 63 and 372 proteins differentially expressed under Pb stress relative to control conditions were identified with liquid chromatography electro spray ionization tandem mass spectrometry in BM and Y1, respectively; with each of these proteins being classified into 14 categories. Hemp adapted to Pb stress by: accelerating adenosine triphosphate (ATP) metabolism; enhancing respiration, light absorption and light energy transfer; promoting assimilation of intercellular nitrogen (N) and carbon (C); eliminating reactive oxygen species; regulating stomatal development and closure; improving exchange of water and CO2 in leaves; promoting intercellular transport; preventing aggregation of unfolded proteins; degrading misfolded proteins; and increasing the transmembrane transport of ATP in chloroplasts. Our results provide an important reference protein and gene information for future molecular studies into the resistance and accumulation of Pb in hemp.
1. Introduction
The heavy metal lead (Pb) is a significant threat to human health, agricultural production and ecological safety. Soil Pb levels have been steadily increasing as a result of human activities and it is now one of the most serious heavy metal pollutants. Pb pollution can persist for more than 300 years with very slow degradation and it can accumulate in soil and living organisms [1]. After absortion by humans, 90% of Pb remains in the skeleton and it can be transferred to the next generation through pregnancy and lactation [2]. Pb has a substantial negative impact on the nervous system, intellectual development, renal function and physical development of infants and children. Mild Pb load can cause neurophysiological damage [2] but severe Pb toxicity leads to genetic mutations and carcinogenesis, which are irreversible [3]. Therefore, the remediation of Pb pollution in the environment is one of the priorities of current studies.
Pb is not required for normal plant physiology and Pb accumulation is usually toxic. Most plants are susceptible to Pb pollution and only a few can tolerate Pb accumulation, including members of the Brassicaceae, Caryophyllaceae, Poaceae, Violaceae and Fabaceae families [4,5,6] but such Pb-accumulators usually exhibit slow growth and low biomass [7]. How to develop Pb-tolerant plants with a high economic benefit, large biomass and rapid growth is an urgent question to be resolved [8]. One feasible approach is the development of new plant varieties with high Pb-accumulation capacity by identifying genes associated with Pb uptake and tolerance using molecular technology and other biotechnologies. Several proteins have been demonstrated to play roles in Pb uptake and tolerance, including NtCBP4 (Nicotiana tabacum calmodulin- binding protein) [9], YCF1 (a cadmium factor in yeast) [10] and TaPCS1 (the low-affinity cation transporter 1 in Triticum aestivum) [11]. Further investigations are needed to identify more related proteins and genes associated with Pb uptake, accumulation or tolerance.
Because of its wide distribution, well-developed root system, rapid growth and high economic value, Industrial hemp (Cannabis sativa L.) plays a significant role in the textile, manufacturing and food industries [12]. Hemp shows a strong Pb tolerance and accumulation ability and it is listed as one of the cultivated crops with Pb tolerance, strong adaptability and high biomass. Hemp can normally germinate in the presence of 1 g/L Pb, where its germination rate is >80% [13,14,15,16]. In addition, hemp can grow in soil with a high level of Pb pollution and it can tolerate Pb accumulation in surface tissues to a level of 26.3 kg/hm2 [14,15,16]. The mechanism of Pb uptake, accumulation and tolerance in hemp has not been reported and the molecular mechanism of Pb tolerance and accumulation is still not clear. Thus, there is a need to investigate the proteins and metabolic pathways associated with Pb tolerance and accumulation. One strategy could be to use proteomics to study the physiological process of adaptation to Pb stress in hemp.
In this study, we used high-throughput proteomics technology to analyze the proteins and metabolic pathways, particularly photosynthesis, energy metabolism and stress response, related to Pb stress in two hemp varieties (one Pb tolerant and one Pb sensitive). These data will provide important molecular reference information for future investigations into Pb tolerance and accumulation in hemp.
2. Materials and Methods
2.1. Cultivation
We used the seed-type hemp variety Bamahuoma (BM) and fiber-type hemp variety Yunma 1 (Y1). While each variety exhibits some Pb tolerance, BM exhibits greater tolerance than Y1, so BM and Y1 were referred to as Pb tolerant and Pb sensitive, respectively. Y1 and BM seeds were kindly provided by the Industrial Corps Institute of Yunnan Academy of Agricultural Sciences and the Guangxi Academy of Agricultural Sciences, respectively.
The pot method was used with a pot height of 24 cm, diameter of 18 cm and holes at the bottom. Each pot was filled with 12 kg bulk material (red soil: humus soil = 1:1 with 17% moisture content) that contained no Pb. Two treatments were performed for each variety: Pb stress, in which Pb(NO3)2 was applied at 3 g/kg soil dry weight and a control, without Pb treatment. Soil was mixed well with the Pb, put into the pots, watered and allowed to sit for 1 week before planting. Fifteen replicate pots were prepared for each variety/treatment combination and each pot contained six hemp plants. A total of 60 pots were placed in a green house and normal management practices were applied.
2.2. Determination of Physiological and Biochemical Indexes
Obvious Pb-toxicity symptoms were observed in both varieties when the average height of the control plants was ~40 cm on average (about 40 days after sowing). Measurements were carried out on the third to fifth leaves from the plant base using a Li-6400 Portable Photosynthetic System (LI-COR, Lincoln, NE, USA) for photosynthetic parameters and spectrophotometry for physiological indicators (soluble protein content, chlorophyll content, malondialdehyde content (MDA) and superoxide dismutase (SOD)) [17]. In addition, 2–4 g of the third to fifth fully expanded leaves from the bottom was randomly collected from three different hemp plants in three different pots undergoing each of the samples (2 treatments × 2 specimens), with three biological replicates. The 12 samples were immediately snap-frozen and stored at −80 °C prior to protein extraction.
2.3. Protein Preparation
Each of the 12 samples was ground to powder in liquid nitrogen with a mortar and pestle, then dissolved with the lysis buffer (7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 40 mM Tris-HCl, 1 mM phenylmethylsulfonyl fluoride, 10 mM dithiothreitol) by sonication for 10 min and centrifuged at 25,000× g for 15 min at 4 °C. The supernatant was precipitated with four volumes of prechilled 10% trichloroacetic acid (TCA)-acetone at −20 °C overnight and centrifuged at 25,000× g for 30 min at 4 °C. The precipitate was washed with prechilled acetone three times. The protein was dissolved with the lysis buffer and protein concentration was determined by the Bradford assay. The samples were reduced with 10 mM DTT for 1 h at 56 °C and alkylated with 55 mM iodoacetamide for 45 min at room temperature in darkness. 100 μg proteins for each sample were digested by the filter-aided sample preparation method with trypsin (Promega, Madison, WI, USA), with 0.1 mM triethylammonium bicarbonate as the buffer solution [18]. Ten μg peptides each sample were pooled and vacuum-dried for building the library.
2.4. Fractionation by High pH Reverse Phase Chromatography
For high pH reverse phase chromatography using the LC-20AB HPLC Pump system (Shimadzu, Kyoto, Japan), the pooled peptides were reconstituted with 0.1 mL buffer A (20 mM ammonium formate, 2% acetonitrile (ACN), pH 10) and loaded onto a Gemini-NX 5 μ c18 110A 150 × 4.6 mm column (Phenomenex, Guangzhou, China). The peptides were eluted at a flow rate of 1 mL/min with a gradient of buffer A for 10 min, 5–30% buffer B (20 mM ammonium formate (FA), 98%ACN, pH 10) for 20 min and 30–80% buffer B for 2 min. Elution was monitored by measuring absorbance at 214 nm and fractions were collected every 1 min. The eluted peptides were pooled as 6 fractions and vacuum-dried.
2.5. LC-ESI-MS/MS Analysis
Each fraction was resuspended in buffer A (2% ACN, 0.1%FA) and centrifuged at 20,000× g for 10 min and 2 μg of peptide was loaded onto nanoLC 425 system (Eksigent part of SCIEX Dublin, CA, USA) with an analytical C18 column (inner diameter 75 μm) packed in-house. The gradient (with a flow rate of 300 nL/min) was set up as follows: 5–20% solvent B for 45 min, 20–24% solvent B for 10 min; 24–30% solvent B for 15 min; 30–35% solvent B for 15 min; 35–90% solvent B for 15 min. Data-dependent acquisition (DDA) was performed with a TripleTOF 5600 System (SCIEX, Concord, ON, Canada). The source parameters were set up as follows: ion spray voltage at 2.3 kV, curtain gas at 30 PSI, ion source gas at 20 PSI and an interface heater temperature of 150 °C. Mass spectrometry (MS) was performed in a high-resolution mode (>30,000 fwhm) for TOF (Time of Flight) MS scans. For the DDA model, the MS range was 350 to 1250 Da with ion accumulation time of 250 ms. Tandem MASS spectrometry (MS/MS) data were acquired in 50 ms and as many as 40 product ion scans were collected if they exceeded a threshold of 125 counts per second (counts/s) and with a 2+ to 4+ charge-state. Dynamic exclusion was set for 1/2 of peak width (15 s). For the sequential window acquisition of all theoretical mass spectra (SWATH) model, the MS range was 350 to 1250 Da with an iron accumulation time of 50 ms, with 57 fixed windows used at 50 ms per SWATH scan with high sensitivity mode with resolution 15 K for m/z 100–1250. All MS data is uploaded to the iProX (https://www.iprox.org, IPX0001592000).
2.6. Library Generation and SWATH Data File Process
Data generated by DDA were searched by Protein Pilot 5.0 (AB SCIEX) with the Paragon algorithm against the hemp transcriptome database (http://genome.ccbr.utoronto.ca/downloads.html, finola1_transcriptome-full.fa.gz). A false discovery rate (FDR) analysis was performed. The output group file was used as the reference spectral library. Then SWATH analysis was processed with the SWATH Acquisition MicroApp 2.0 in PeakView® Software (SCIEX, Foster City, CA, USA) with the following parameters: five peptides/protein, five transitions/peptide, peptide confidence level of > 90%, FDR < 1%, excluded shared peptides and Ion Library Mass Tolerance of 100 ppm. The quantitation data was normalized with the median. Three biological replicates for each sample were performed for t-test analysis. The protein levels with a p-value less than 0.05 and ratio ≥1.5 (increase) or ≤0.67 (decrease) were considered significantly different in our experiment.
3. Results and Discussion
3.1. Physiological Indicators under Pb Stress
The growth stage at which the plants were sampled was selected to coincide with rapid growth (40d after sowing). The indicators of Pb stress in BM were more stable than those in Y1, especially the net photosynthetic rate and MDA level (Table 1), indicating that the Pb tolerance of BM was higher than that of Y1 during rapid growth, which can also be verified in the hemp growth (Figure 1).
Pb stress affected all the photosynthetic indicators in hemp. The net photosynthetic rate (Photo), stomatal conductance (Cond) and transpiration rate (Tromol) of Y1 under Pb stress were reduced by 19%, 12.8% and 14.4%, respectively, compared to the controls and the decrease in net photosynthetic rate was significant. However, the intracellular CO2 (Ci) concentration was increased by 5% under Pb stress. In BM, only the net photosynthetic rate was decreased by 4.7% due to Pb stress and all other indicators were higher than those in the controls. Among these, the Ci concentration was significantly elevated by 10.2%. Thus, Pb-tolerant BM exhibited a reduction (albeit much less than in Y1) in photosynthetic rate upon Pb treatment but it could adapt to the stress by increasing the Ci, Cond and Tromol values.
The effects of Pb stress on soluble protein content, chlorophyll, SOD and MDA showed that the first three indicators decreased under Pb stress in both BM and Y1 but the MDA content of Y1 was significantly elevated by 62.3% under Pb stress, whereas only a 4% non-significant increase was observed in BM. MDA is one of the products of membrane lipid peroxidation, so higher MDA suggests greater damage to the membranes. Therefore, it was revealed that Pb stress resulted in greater damage to the membrane system of Y1 than that of BM; that is, the membrane system of Y1 was more susceptible to Pb stress.
3.2. Protein Identification and Analysis
A total of 2131 proteins were identified in the SwissProt/UniProt database (Table S1) and 63 and 372 were recognized as being differentially expressed (≥1.5-fold) under Pb stress compared to the control conditions in BM (Table S2) and Y1 (Table S3), respectively. Of these, 39 and 231 proteins in BM and Y1, respectively, were upregulated under Pb stress, which accounted for 61.9% and 62.1% of the differentially expressed proteins in BM and Y1, respectively, suggesting that the metabolic status and molecular physiological activities of the Pb-tolerant variety BM were more stable than those of the Pb-sensitive variety Y1 under Pb stress and the high metabolic activity in Y1 resulted in Pb sensitivity.
The different proteins in each cultivar were functionally classified according to the method proposed by Bevan et al. (1998) [19] (Figure 2 and Figure 3). They were classified into 14 categories: primary metabolism, energy, protein destination and storage, disease/defense, protein synthesis, photosynthesis, transport, transcription, signal transduction, cell structure, secondary metabolism, intracellular traffic, cell growth/division and unknown. Over 67% of proteins associated with protein destination, storage and transcription were downregulated in the differentially expressed proteins of Y1, whereas >91% of proteins associated with transporters and photosynthesis in this variety were upregulated. In BM, proteins associated with transporters, protein synthesis, energy and secondary metabolism were upregulated under Pb stress, while proteins related to cell growth/division, intracellular traffic and photosynthesis were downregulated (Figure 2 and Figure 3).
4. Conclusions
We used high-throughput SWATH technology to investigate the tolerance mechanisms of Pb-tolerant BM and Pb-sensitive Y1 under Pb stress. There were 63 and 371 differentially expressed proteins in Pb-stressed BM and Y1, respectively, which were classified into 14 categories. The following responsive mechanisms are used by hemp to cope with Pb stress: (1) Increasing ATP biosynthesis; (2) enhancing respiration; (3) strengthening the connection between respiration and photosynthesis; (4) improving photosynthesis; (5) promoting N and C assimilation; (6) eliminating reactive oxygen species; (7) regulating stomatal development and closure to increase air exchange; (8) elevating water transportation; (9) shortening the growth period and controlling flowering time; (10) protecting unfolded proteins from aggregation and degrading misfolded proteins; and (11) boosting the transmembrane transport of ATP and within chloroplasts. Our results provide important reference information on the proteins involved in Pb tolerance in hemp for future studies.
In this study, we used SWATH technology to study the difference in proteomics between the leaves of Pb-tolerant seed-type hemp variety BM and the Pb-sensitive fiber-type hemp variety Y1 under Pb stress. The response mechanism of BM to Pb stress and the important proteins related to the response mechanism were obtained. However, the important proteins obtained in this study have not undergone subsequent functional verification. Therefore in subsequent studies, it is still necessary to verify the function of these proteins by using the gene cloning technology in the future research.
Appendix Group
Supplementary Materials
The following are available online at https://www.mdpi.com/2073-4425/10/5/396/s1, Table S1: List of hemp proteins identified in this study. Three control samples of Y1 were labeled with tags YD1, YD2 and YD3, three Pb-treatment samples of Y1 were labeled with tags YC1, YC2 and YC3, three control samples of BM were labeled with tags BD1, BD2 and BD3, three Pb-treatment samples of BM were labeled with tags BC1, BC2 and BC3. Table S2: List of differentially expressed proteins in BM leave under Pb stress (control and Pb-stress samples were labeled with BD1, BD2, BD3 and BC1, BC2, BC3 tags, respectively). Table S3: List of differentially expressed proteins in Y1 leave under Pb stress (control and Pb-stress samples were labeled with YD1, YD2, YD3 and YC1, YC2, YC3 tags, respectively).
Funding
This research was supported by a grant from the National Natural Science Foundation of China (31501350, 31660351 and 31760403), National Natural Science Foundation of Yunnan (2016FB068 and 2017FD060) and China Agriculture Research System (CARS-16-E15).
Conflicts of Interest
The authors declare no conflict of interest.
| Treatment | Photo (μmolm−2·s−1) | Cond (mmol·m−2·s−1) | Ci (μl·L−1) | Tromol (g·m−2·h−1) | Chlorophyll (mg/g) | Soluble Sugar (mg/g) | SOD (U/g) | MDA (nmol/g) | |
|---|---|---|---|---|---|---|---|---|---|
| BM | Pb | 14.3 ± 0.46 | 0.51 ± 0.02 | 342.00 ± 6.00 * | 9.54 ± 0.29 | 68.31 ± 0.06 | 295.14 ± 0.65 | 193.12 ± 5.63 | 0.98 ± 0.38 |
| CK | 15.0 ± 0.50 | 0.43 ± 0.09 | 310.33 ± 9.87 | 8.99 ± 0.32 | 68.75 ± 0.03 * | 304.13 ± 0.42 * | 195.00 ± 1.88 | 0.94 ± 0.17 | |
| IR | -4.7% | 17.6% | 10.2% | 6.1% | -0.6% | -3.0% | -1.0% | 4.0% | |
| Y1 | Pb | 14.03 ± 1.00 | 0.48 ± 0.02 | 322.67 ± 4.16 * | 8.24 ± 0.53 | 68.51 ± 0.11 | 288.47 ± 1.64 | 200.62 ± 3.75 | 0.86 ± 0.19 * |
| CK | 17.33 ± 1.14 * | 0.55 ± 0.05 | 306.00 ± 2.65 | 9.74 ± 0.26 * | 68.92 ± 0.10 * | 292.94 ± 4.25 | 203.12 ± 9.44 | 0.53 ± 0.08 | |
| IR | −19.0% | −12.8% | 5.4% | −14.4% | −0.6% | −1.5% | −1.2% | 62.3% | |