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Influence of organic substrates on nutrient accumulation and proteome changes in tomato-roots
Scientia Horticulturae 252 (2019) 192–200
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Influence of organic substrates on nutrient accumulation and proteome changes in tomato-roots Jiayi Xinga,b, Nazim Grudac, Jing Xionga, Wei Liua,b,
T
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a
Vegetable Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing, 100097, China Key Laboratory of Urban Agriculture (North China) by Ministry of Agriculture and Rural Affairs, Beijing, 100097, China c INRES – Horticultural Science, University of Bonn, 53121, Bonn, Germany b
A R T I C LE I N FO
A B S T R A C T
Keywords: Organic growing media Soilless culture Coir Peat-vermiculite substrate Nutrient accumulation Proteome
The gradually accumulation of nutrients in organic substrates during growing season may have adverse effects on plant development. This study was conducted to determine the mode of nutrient accumulation and alteration at molecular level induced by the excess nutrient stress in tomato. The ICP-MS was used to analyze mineral ions in root zone solution and nutrients uptake by tomato plants; and iTRAQ-based proteomic analysis was conducted to identify proteome changes in tomato roots. Coir and peat-vermiculite were used in substrate treated experiments. Results showed that the content of K+ was higher in coir than in peat-vermiculite whereas it was the opposite for Ca2+. The concentrations of NO3−, SO42- and Mg2+ were significantly higher in peat-vermiculite and coir substrates than in water culture. The peat-vermiculite substrates generally enhanced Ca uptake but reduced P uptake by plants, when compared to both coir and water culture. Compared to water culture, functional annotation analysis of the root proteome revealed that the excess nutrient accumulation induced complex proteomic alterations involved in mineral ion binding and transport. A total of 358 differentially abundant proteins (DAPS) were identified, including 11 mineral ion binding and transport related proteins, such as calmodulin-like protein and nitrate transporter 3.2 under peat-vermiculite and coir cultivations. RT-qPCR was used to validate nine genes encoding DAPS. We believe that these indicators will contribute to a better control of soilless culture systems and a waste reduction in production of tomatoes.
1. Introduction Organic substrates, such as peat and coir, are widely used in vegetable crop production around the world (Gruda, 2012; Savvas and Gruda, 2018). The physical properties of organic substrates depend on the materials and containers used, growing media (GM) compression or density, i.e. the amount of substrate in a certain volume, the applied irrigation system and root development (Gruda and Schnitzler, 2004). According to Gruda and Schnitzler (2004), under certain conditions, organic GM may have a limited air and low water buffer capacity, sometimes accompanied with failures and a bottleneck situation of nutrients (Gruda and Schnitzler, 1999). During the cultivation period, since nutrient supply and crop fertilizer absorption are not completely consistent, some mineral elements may gradually accumulate in the organic substrates around the root zone (Savvas and Gizas, 2002). Nutrient accumulation in the root zone can negatively influence crop growth through inducing osmotic stress in the rhizosphere and then limiting the absorption of water and nutrients by roots (Gruda, 2009).
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Moreover, it may lead to nutrient imbalance and ion toxicity in crops (Carmona et al., 2012; Urrestarazu et al., 2008), further resulting in yield decline and quality deterioration (Gruda and Schnitzler, 2004; Changa and Lee, 2016). In recent years, proteomics has been widely used to identify salt tolerance proteins in cucumber and other vegetable crops (Fan et al., 2015; Li et al., 2015; Jiang et al., 2017). Isobaric tags for relative and absolute quantitation (iTRAQ), a mass spectrometry based quantitative method, which can identify and quantify proteins of multiple high coverage samples at the same time (Wiese et al., 2007). To date, this method has been successfully applied in many studies related to a variety of abiotic stress conditions, because of its high sensitivity and more accurate quantification (Chen et al., 2016; Liu et al., 2017; Jiang et al., 2017). Changes in root proteomes is one of the most direct signals and metabolic factors that can be easily induced by suboptimal conditions in the root zone (Zhang et al., 2012). Therefore, investigating the protein changes in response to different cultivation substrates may help to reveal the potential mechanism of nutrient accumulation in the root
Corresponding author at: Vegetable Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing, 100097, China. E-mail address: [email protected] (W. Liu).
https://doi.org/10.1016/j.scienta.2019.03.054 Received 14 September 2018; Received in revised form 12 February 2019; Accepted 23 March 2019 0304-4238/ © 2019 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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zone (Asaduzzaman et al., 2013). Tomato is the most cultivated crop worldwide. The soilless culture system for growing tomatoes utilizes organic materials (e.g. peat and coir) as GM rather than rockwool. Nutrient accumulation in the root zone is a major limiting factor that could adversely affect plant growth (Xiong et al., 2017). Although previous studies have investigated the influences of different organic substrates on the retention and availability of nutrients in root zone, it is still unclear how organic substrates affect root protein changes under nutrient accumulation conditions (Sun et al., 2016). To meet this need, we investigated the characteristics of nutrient accumulation in tomato root zone under coir cultivation (CC) and peat-vermiculite cultivation (PV), while using water culture (WC) as control. Moreover, we used iTRAQ analysis to comprehensively assess dynamic changes of proteome in tomato roots under different cultivation systems (i.e. CC, PV and WC). The aims of this study were to investigate the expression and regulation mechanism of main ion channel proteins during nutrient accumulation process and to understand how tomato roots respond at protein levels to nutrient accumulation in organic GM.
Table 2 Nutrient formula of nutrient solution. Macroelement
Concentration (mmol L−1)
Microelement
Concentration (μmol L−1)
NO3− NH4+ H2PO4− K+ Ca2+ Mg2+ SO42−
15.4 1.4 1.8 9.3 3.9 1.4 2.1
Fe Mn Cu Zn B Mo –
14.7 27.8 0.8 6.7 4.2 0.07 –
2.3. Nutrient solution management A closed-loop soilless culture system was used. Each gutter was fed from a nutrient solution tank. The nutrient solution drained directly back into the same tank where it was mixed with the new solution. The compositions of standard nutrient solution were shown in Table 2. The electrical conductivity (EC) and pH of the nutrient solution tanks were monitored every week. To maintain the EC of 2.3 dS m−1, the nutrient tank was maintained at 200 L by replenishing with fresh solution containing fresh water, (EC 0.12 dS m−1, pH 7.18; Na+ 0.6 mmol L−1, Ca2+ 0.1 mmol L−1, Mg2+ 0.05 mmol L−1, SO42− 0.2 mmol L−1, NO3− 0.7 mmol L−1, NH4+ 0.05 mmol L−1and H2PO4− 0.02 mmol L−1). The nutrient solution was applied through a drip (average flow rate of 1.5 L h−1) irrigation system with one dripper per plant. The irrigation frequency and volume were the same for all substrate culture gutters. Nutrient solution was supplied four times per day (9:00, 11:00, 13:00, and 15:00) for 10 min each, irrigation volume was 2 L per plant. For all water culture gutters, the nutrient solution was circulated for 30 min. in every two hours from 8:00 am to 6:00 pm. Every 2 months, the nutrient solution tank was washed and the nutrient solution in the tank was completely refreshed.
2. Materials and methods 2.1. Experimental site and crop planting The experiment was conducted in a climate-controlled greenhouse in Beijing Vegetable Research Center, Beijing Academy of Agriculture and Forestry Sciences in Beijing. The average light intensity ranged from 18.3–136.8 μmol m−2 s-1, and the average temperature was at 14.0–23.0℃, respectively. Tomato (Solanum lycopersicum) ‘Lucius F1’ seeds were sown in plug tray and thirty-three days later they were transplanted into coir cubes (10 cm × 10 cm). Thirty-three days after planting in the substrate cube, the tomato seedlings were transplanted into substrate slabs (CC and PV) or nutrient solution (WC). The planting density was 2.4 crops m−2.
2.4. Root-zone solution analysis From 3 weeks after transplanting, root-zone solution was sampled every two weeks. Root-zone solution (100 ml) was collected with a root solution extractor installed between the crops. The samples were stored at 2 °C until further analyzing. The EC was measured using a multi meter (Multi 3420 SET C., WTW, Germany). K+, Ca2+, Mg2+, NO3−and H2PO4− were assayed by inductively coupled plasma spectrometry (ICPE-9000, Shimazu, Japan).
2.2. Experimental design Coir was bought from Jiffy Group in Netherland. Both peat and vermiculite were bought from Beijing Lide Agricultural S&T Development Company in China. The mixture of peat and vermiculite (v/v, 2:1) (PV) were used as cultivation substrates in the experiment. Selected characteristics of different substrates were showed in Table 1. The experiment was a completely randomized block design with three replicates and each replicate contained a cultivation gutter of 10 m long. Tomato crops were planted with 30-cm plant spacing and each gutter had 30 plants. For each substrate culture gutter, 10 substrate slabs (100 cm × 20 cm × 7.5 cm) were installed. For each water culture gutter, semicircular PVC pipe of 32 cm in diameter was used. The depth of the nutrient solution was 10 cm.
2.5. Analysis of root system development 50, 80 and 107 days after transplanting, roots of one representative plant from replicate of all the treatment conditions were sampled. Roots were washed on 0.5 mm diameter sieve, scanned (SNAPSCAN 1236, AGFA, Germany) and then analyzed with the WinRHIZO software package (Regent Instruments Inc. Canada) for root length, diameter and surface area. The roots were further divided into four classes according to their diameters (i.e. 0–0.2, 0.2−0.6, 0.6−0.9, > 0.9 mm).
Table 1 Selected physical and chemical properties of coir and peat-vermiculite. Properties
Coir
Peat-vermiculite
EC (dS m−1) pH C (%) N (mg kg−1) P (mg kg−1) K (mg kg−1) Ca (mg kg−1) Mg (mg kg−1) S (mg kg−1) Total Porosity (%) Bulk density (g cm−3)
0.10 6.1 49.5 44 38 1560 58 55 405 85.6 0.2
1.10 7.1 15.9 64 42 246 1668 636 645 66.0 0.4
2.6. Plant nutrient analysis Similarly to roots analyses, roots, stems, leaves and fruits of one plant from each replicate of all treatment conditions were sampled on days 50, 80 and 107 after transplanting. After washing with distilled water, they were dried in a ventilated oven at 75 °C to constant weight. Nutrient contents in roots, stems, leaves and fruits samples were analyzed. The contents of K, Ca, Mg and P were assayed after digestion with H2SO4-HNO3-HClO4 (H2SO4: HNO3: HClO4 = 1 ml: 5 ml: 1 ml) by inductively coupled plasma spectrometry (ICPE-9000, Shimazu, Japan). 193
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2.7. Protein isolation, digestion, and labeling with iTRAQ reagents On day 107 after transplanting, the whole roots of one plant from each replicate were harvested. Protein extraction of roots was conducted using the phenol extraction/methanol-ammonium acetate precipitation approach (Faurobert et al., 2007). Protein concentration was determined by BCA Protein Quantitation Kit (Sangon Biotech, Shanghai, China). After reduction and cysteine-blocking as described in the iTRAQ protocol (AB Sciex, Concord, ON), solutions containing 100 μg protein were digested overnight at 37 °C with sequence grade modified trypsin (Promega, Madison, WI) and then labeled with different iTRAQ tags as follows: IT113 and IT114 for WC samples; IT117 and IT118 for PV samples; IT119 and IT121 for CC samples. The labeled samples were then pooled and dried on a rotary vacuum concentrator.
Fig. 1. Electrical conductivity (EC) in root-zone solution under water culture (WC), peat-vermiculite (PV) and coir (CC) cultivations. The vertical bars represent the standard errors. Black letter, green letter and purple letter WC, PV and CC cultivations, respectively (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
2.8. LC–MS/MS analysis The peptide mixtures were pressure-loaded onto a fused silica capillary column packed with 3-μm dionex C18 material (RP; Phenomenex). The RP sections with 100 Å were 15 cm long, and the column was washed with buffer A (water, 0.1% formic acid) and then buffer B (ACN (Acetonitrile), 0.1% formic acid). After desalting, a 5mm, 300-μm C18 capture tip was placed in line with an Agilent 1100 quaternary HPLC (High Performance Liquid Chromatography) and analyzed using a 12-step separation.
3. Results 3.1. EC in root-zone solution The EC in root zone solution of all substrates was maintained at relatively stable levels in the first 9 weeks after transplanting, and then increased gradually during the next 6 weeks (Fig. 1). In the 15th week after transplanting, the EC in root-zone solution reached 7.71 dS m−1 in CC, 5.86 dS m−1 in PV, and 4.21 dS m−1 in WC, inferring the serious nutrient accumulation situation in root zone under substrate cultivation systems.
2.9. DAPS data analysis Tandem mass spectra were searched against mascot 2.1 (Local Host) uniprot_solanum_lycopersicum. Fasta. The search results were then filtered using a cutoff of 1% for peptide false identification rate. Peptides with Z score < 4 or Delta-Mass > 5 ppm were rejected. Furthermore, the minimum number of peptides to identify a protein was set to 1. The default parameters for the Quantitative software Profile Analysis 2.0 were used throughout the analysis. Bioinformatics analysis was carried out to categorize proteins based on biological processes, cellular component and molecular function using annotations in Protein Analysis Through Evolutionary Relationships (PANTHER) database v 6.1(www. pantherdb.org), which is in compliance with gene ontology (GO) standards.
3.2. Root length, root surface area and average root diameter for tomato A smaller root surface area was found in WC, when compared to PV and CC (Table 3). On days 80 and 107 after transplanting, both the root length and surface area were larger in WC than in PV and CC. With respect to average root diameter, it was significantly higher in CC than in PV on day 50, and in WC and PV on days 80 and 107 after transplanting. Overall, thin roots (0-0.2 mm diameter) had the highest proportion of total root length under all treatments (Fig. 2). On days 80 and 107 after transplanting, the proportion of thin roots was significantly higher in WC and PV than in CC. However, reverse trends were found in relatively thicker roots (i.e. 0.2−0.6 mm, 0.6−0.9 mm and > 0.9 mm diameter) among treatments. The highest proportion of total surface was generally found in thin roots (0-0.2 mm diameter) under WC and PV, but in relatively thicker roots (> 0.2 mm) under CC (Fig. 3).
2.10. RNA extraction and RT-qPCR 107 days after transplanting, total RNA extraction and reversetranscription quantitative PCR (RT-qPCR) analysis of roots were carried out according to the method as described in Sun (2016). All the primer pairs used for RT-qPCR were designed using the Beacon Designer 7.0 and provided in Table S1. The data were processed with the 2−△△CT method as described by Livak and Schmittgen (2001).
3.3. Ions accumulation in root zone solution The concentration of K+ in root-zone solution was significantly higher in CC than in WC and PV, and in WC than in PV (Table 4). The concentration of Ca2+ in root-zone solution was higher in PV than in CC. With respect to Mg2+, NO3− and Ca2+, the ion concentrations were higher in PV and CC than in WC. Moreover, the concentration of H2PO4− in root-zone solution was significantly higher in CC than in WC and PV.
2.11. Fruit yield During fruit ripening period, for each cultivation gutter, fruits were harvested from 18 plants from each replicate to measure fresh yield. At the end of the cropping season, the fresh yield of each harvest was summed up as the total yield (t hm−2).
3.4. Nutrient distribution in tomato plants and fruit yield 2.12. Statistical analysis As shown in Table 5, the investigated systems generally had different effects on the nutrient distribution among different plant organs. In roots, only the concentration of Ca was significantly influenced and increased by PV compared to WC and CC. In both stem and leaf, the concentration of P was significantly lower in PV than in WC and CC, but
Statistical analysis was carried out with SPSS 20.0 software (SPSS statistical package, Chicago, IL, USA). Multiple comparisons using Duncan’s test were done whenever the ANOVA indicated significant differences (P ≤ 0.05). 194
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Table 3 The dynamics of root length, root surface area and average root diameter for tomato under water culture (WC), peat-vermiculite (PV) and coir (CC) cultivations. Treatment
WC PV CC a
Root surface area (m2)
Root length (m)
Average root diameter (mm)
50 d
80d
107 d
50 d
80d
107 d
50 d
80d
107 d
338.5 a 434.7 a 399.2 a
1361.2 a 901.2 b 757.2 b
1556.2a 1251.9ab 839.3 b
0.25 b 0.37 a 0.35 a
1.14 a 0.79 b 0.73 b
1.10 a 0.89ab 0.73 b
0.24ab 0.22 b 0.28 a
0.22 b 0.24 a 0.31 a
0.23 b 0.21 b 0.28 a
The same letter denotes no significant difference among different treatments (LSD test at P < 0.05).
accumulation stress reaction. Moreover, some DAPS were found to be remarkably enriched in stress-response-related biological processes, such as “response to oxidative stress” and “response to biotic stimulus”. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis (p < 0.05), the DAPS were mainly enriched into plant biology processes, including ribosome, phenylpropanoid biosynthesis, carbon metabolism, chemical carcinogenesis, protein processing in endoplasmic reticulum, drug metabolism - cytochrome P450, metabolism of xenobiotics by cytochrome P450, starch and sucrose metabolism and glutathione metabolism (Fig. S2).
a reverse trend was found in the concentration of ca. Additionally, the concentrations of Mg in both stem and leaf were significantly higher in PV and CC than in WC. No significant difference in nutrient concentration was generally found in the fruit among treatments. For fruit yields only the first two trusses were recorded and no significant differences were found between the treatments. 3.5. Identification of DAPs using iTRAQ LC–MS/MS A total of 21,284 peptides and 4163 non-redundant protein groups were identified in tomato roots after 15 weeks of transplanting, and the labeling efficiency was 98.24%. To obtain differentially abundant proteins (DAPs), proteins were compared between each pair of treatments (WC, CC and PV). The proteins with ratio values of more than 1.2 or less than 0.83 (P < 0.05) were defined as DAPs. A total of 358 DAPs in the above 3 comparison schemes were identified in all 3 replicates. Based on this criteria, 128, 51, and 154 proteins were identified to be up-accumulated, and 83, 32, and 204 proteins were down-accumulated during CC vs. WC, CC vs. PV, and WC vs. PV, respectively (Fig. 4). GO annotations (GO) indicated that the 358 identified proteins were involved in 30 biological processes, 25 cellular components and 30 molecular functions (Figs. 5 and S1). Among these categories, “oxidation-reduction process”, “metabolic process”, “response to oxidative process”, “hydrogen peroxide catabolic process” and “carbohydrate metabolic process” were the most common biological processes. In cellular component category, “extracellular region”, “integral component of membrane” and “membrane” were the central categories. “The metal ion binding”, “oxidoreductase activity” and “hydrolase activity” were the most dominant categories in molecular function. Several important cellular components were closely related to the transport activities of water and various ions such as “plasma membrane” and “proton-transporting ATP synthase complex, catalytic core F(1)”. Further analysis identified a few of DAPS that were closely correlated with “nitrate transport,” “metal ion binding” and “zinc ion binding”, suggesting that those ion channels might play an important role in nutrient
3.6. Validation of DAPs by RT-qPCR The expression of nine genes, which were associated with ion absorption and transport-related proteins (SlCML1, SlNTR3.2, SlMscL10, SlPCaP1 and SlTMP1), carbohydrate metabolism (SlCA3), auxin-activated signaling pathway signaling pathway (SlARF18), response to oxidative stress (SlCAT3) and stress and defense (SlPHOS32), were determined by RT-qPCR to confirm the relationships between the expression changes of RNA and corresponding proteins (Fig. 6; Table 4). A majority of the nine genes showed a matching abundance level from mRNA to protein when the plants were treated under different substrates. However, the genes encoding carbonic anhydrase3 (SlCA3) and plasma membrane-associated cation-binding protein 1 (SlPCaP1) were inversely correlated between the mRNA and protein changes. 4. Discussion Currently, the soilless culture systems are regarded as a major technological component of modern greenhouses (Grunert et al., 2016). The independence from the soil as a rooting medium enables optimization of both physical and chemical characteristics in the root environment and a more efficient control of pathogens without the need to apply soil fumigation (Gruda, 2012). Both water culture systems with merely nutrient solution as root environment and cultivation on porous
Fig. 2. Ratio of root length with certain diameter to total root length for tomato under water culture (WC), peat-vermiculite (PV) and coir (CC) cultivations. The vertical bars represent the standard errors. Different letters indicate significant difference between treatments according LSD test at P < 0.05. 195
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Fig. 3. Ratio of root surface area with certain diameter to total root surface area for tomato under water culture (WC), peat-vermiculite (PV) and coir (CC) cultivations. The vertical bars represent the standard errors. Different letters indicate significant difference between treatments according LSD test at P < 0.05.
growing media which create a matrix that can retain both air and water at suitable ratios for plant growth included in these systems (Savvas and Gruda, 2018). In this study we used a peat-vermiculite, a coir and water culture system, in order to investigate the expression and regulation mechanism of main ion channel proteins and the response of tomato roots at protein levels to nutrient accumulation in soilless culture systems. The root is the main organ of a plant to absorb water and nutrient. The root epidermis is the region with the highest absorption activity, and the increase of root length could improve the efficiency of water and nutrient use (Rich et al., 2011). Therefore, the root surface area and root length are the key indicators to measure root absorption capacity. In this study, WC provided a relatively normal environment for root growth compared to substrate cultures according to the root zone EC (Fig. 1). The EC in root zone solution of substrate treatments was maintained at relatively stable levels during the first 9 weeks after transplanting and increased much faster than WC thereafter. Correspondingly, there was no significant difference in root length among treatments 50 days after transplanting, while both root length and root surface area of PV and CC were significantly lower than those of WC 80 days after transplanting (Table 3). It can be postulated that as plants are exploring for nutrients, a larger root system should be developed. Gruda reported a higher root development with longer roots in wood fiber substrates with a lower level of nitrogen supply as in good balanced nitrogen treatment (Gruda et al., 2012). CC had an even higher root zone EC than that of PV, where root length and root surface area of CC were also lower than that of PV. The results indicated that nutrient accumulation in substrate affected the root growth, especially for thin roots (0–0.2 mm diameter) which had the largest proportion of total length and surface area (Figs. 2 and 3). From the beginning, average root diameter of CC was higher than that of PV and WC. During the whole study period, the average root diameter of each treatment kept the same. These results suggested that the average root diameter was mainly affected by the physical properties of substrates (Kato et al., 2010). Nutrient accumulation in root zone is critical for plant growth under substrate cultivation (Changa and Lee, 2016). For all substrates, most
Table 5 Nutrient distribution in tomato plants, cultivated in water culture (WC), peatvermiculite (PV) and coir (CC) cultivations. Organs
Root
Stem
Leaf
Fruit
Treatment
WC PV CC WC PV CC WC PV CC WC PV CC
Nutrient (mg/g dry matter) P
K
Ca
Mg
2.2 a 2.2 a 2.4 a 9.4 a 6.1 b 9.5 a 11.5 a 6.3 b 10.9 a 6.1 a 5.3 a 6.2 a
3.3 a 3.3 a 4.1 a 127.4 128.5 144.6 143.3 120.8 127.9 106.5 110.0 119.1
32.4 b 37.1 a 34.1 b 25.7 b 31.5 a 25.9 b 83.5 b 91.4 a 79.3 b 1.1 a 1.8 a 2.0 a
3.7 a 3.2 a 4.0 a 5.2 b 7.5 a 7.5 a 10.5 b 13.4 a 14.7 a 3.5 a 3.9 a 3.9 a
a a a a a a a a a
All data were average value of seven sampling times. a The same letter denotes no significant difference among different treatments (LSD test at P < 0.05).
mineral ions increase gradually as the growing time extended, resulting in gradually increased EC in root zone (Xiong et al., 2017). Organic substrates release certain kinds of ions to solution meanwhile absorb other ions. In this study, we noted that in the root zone of plants cultivated in coir a relatively high K+ content were accumulated, while peat-vermiculite had a high Ca2+ content. The concentration of K+ in root zone solution was significantly higher in CC than in PV and WC, and Ca2+ content was higher in PV than in CC and WC (Table 1, Table 3), probably due to that CC released K+ while PV adsorbed K+ to and from solution, respectively (Barrett et al., 2016; Rippy and Nelson, 2007). Compared with WC, the concentrations of NO3−, SO4− and Mg2+ were significantly higher in the root zone of organic substrates CC and PVC (Table 4), and this might be an important factor leading to the nutrient accumulation stress (Fig. 1). The concentration of Ca2+ in root zone solution was higher in PV than in CC and WC. The Ca2+ concentration in root, stem and leaf of PV was also significantly higher
Table 4 Some nutrient in root zone solution under water culture (WC), peat-vermiculite (PV) and coir (CC) cultivations. Treatment
K+ mmol L−1
Ca2+ mmol L−1
Mg2+ mmol L−1
NO3− mmol L−1
SO4− mmol L−1
H2PO4− mmol L−1
WC PV CC
11.43 b 7.45 c 18.07 a
5.62 ab 9.03 a 4.35 b
2.18 b 3.77 a 3.66 a
12.65 b 54.32 a 59.61 a
1.95 b 7.58 a 8.57 a
1.95 b 2.58 b 5.57 a
All data are average value of seven sampling times. a The same letter denotes no significant difference among different treatments (LSD test at P < 0.05). 196
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used to understand the molecular response mechanisms of tomato root under nutrient accumulation stress, and a total of 358 were DAPs identified. Among them, a number of proteins that regulate the binding and transport of ions were identified with altered abundance under treatment conditions (Table 6). Calcium signaling pathways play an important role in triggering complex stress responses in plants (Grant et al., 2015). Calmodulin (CaM) and calmodulin-like proteins (CMLs) are major Ca2+ sensors, playing critical roles in interpreting encrypted Ca2+ signals under stress.(Wang et al., 2015) AtCML8 and CML37 were found to be induced by salt treatment (Park et al., 2010; Vanderbeld and Snedden, 2007). CML1 is an important Ca2+ signaling sensor, and it has been shown to be involved in many responses to salt stress (Srivastava et al., 2013). This protein was significantly up-accumulated in CC and PV compared to WC, indicating that calcium signaling pathway plays an essential and positive role in tomato response to nutrient accumulation stress under organic substrate cultivation. K+ is an essential ion for many physiological processes, especially for enzyme activation and protein synthesis under stress (Anschütz et al., 2014). In our data, five H+-ATPase activity related protein, including three F0F1type H+-ATP synthase delta subunits, FtsH protease and ABC transporter E family member 2-like, were up-accumulated in PV, compared to WC and CC. F0F1-type H+-ATP could affect the transport of K+ in cotton under salt stress (Gong et al., 2017), and we hypothesized that it may be a key factor leading to the lower concentration of K+ in PV, in addition to a part of K+ absorbed by PV (Xiong et al., 2017). The concentration of NO3− in root-zone solution was the highest of all ions, and it is also the largest contribution to nutrient accumulation stress. Nitrate transporters (NTR) are induced by nitrate, and can make plants grow normally under various nitrogen concentrations (Kiba et al., 2012). High-affinity nitrate transporter 3.2-like protein had a highest expression in WC, and this may explained why the concentration of NO3- was the lowest in WC. It shows that increasing the expression of high-affinity nitrate transporter 3.2-like protein can enhance plant resistance nutrient accumulation stress. We identified several additional proteins related to mineral ion binding, including hyoscyamine 6-dioxygenase, purple acid phosphatase (PAPs), polyphenol oxidase (PPO) F, putative copper ion bindinglike and putative ADP-ribosylation factor GTPase-activating protein AGD9-like. PGPAP18 may play a defensive role against environmental
Fig. 4. Identification and statistics of differential abundance protein species (DAPS) under water culture (WC), peat-vermiculite (PV) and coir (CC) cultivations. (A) Number of up- or down-accumulated protein species between two different cultivations. (B) Venn diagram analysis of up-accumulated and downaccumulated protein species.
compared to WC and CC. Accumulation of mineral ions could lead to an increase in EC value, resulting in nutrient accumulation stress in root zone of organic substrate crops (Barrett et al., 2016). An iTRAQ-based quantitative was
Fig. 5. The most significantly-enriched gene ontology (GO) terms of differential abundance protein species (DAPS) under water culture (WC), peat-vermiculite (PC) and coir (CC) cultivations. 197
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Fig. 6. Relative mRNA expression analysis using RT-qPCR on nine protein species under water culture (WC), peat-vermiculite (PV) and coir (CC) cultivations. The expression level under WC was set to a value of 1. Each bar shows the mean ± SE (n = 3). Letters above the columns indicate significant differences at p < 0.05 according to Duncan’s multiple range test. Nine protein species were auxin response factor 18-like (ARF18), carbonic anhydrase3 (CA3), calmodulin-like protein 1 (CML1), mechanosensitive ion channel protein 10 (MscL10), high-affinity nitrate transporter 3.2-like (NTR3.2), plasma membrane-associated cation-binding protein 1-like (PCaP1), suberization-associated anionic peroxidase 1-like (TMP1), universal stress protein PHOS32 and Catalase-3 (CAT 3).
5. Conclusion
stress and endow stress-tolerant plant with stress adaptation. (Reddy et al., 2017). PPOs are Zn- and Cu-responsive and involved in metal ionassociated gene networks in Populus (Lu et al., 2011), and overexpression of a potato PPO cDNA could improve the disease resistance in tomato (Li et al., 2002). These proteins were all up-accumulated in CC and PV, compared to WC, implying that mineral ion binding pathway might be managed by complex regulatory mechanism to cope with nutrient accumulation stress in tomato. The generation of ROS is one of the main stress-induced universal consequences in plants (Meloni et al., 2003; Guo et al., 2012). It can not only cause irreversible damage to cells, but also attack macromolecules. A series of antioxidants and enzymes are needed to scavenge ROS in plants (Sun et al., 2017). In this study, we identified a total of 26 DAPs related to oxidative stress response (Table 6). Among those DAPs, peroxidase and catalase were two major categories, most of which were up-accumulated in organic substrates (CC and PV) compared to water culture (WC), suggesting that their significant roles in response to nutrient accumulation stress. Additionally, a putative universal stress protein A-like, another protein species related to stress tolerance, was also identified (Table 6). This protein has been demonstrated with functions in enhancing plant’s resistance to unfavorable conditions through forming a natural biological defense mechanism (Tkaczuk et al., 2013). The expression of putative universal stress protein A-like was markedly higher in CC than in PV and WC, indicating that tomato plants expressed a more active response to the stress conditions on the CC substrate.
Compared with water culture, organic substrates (PV and CC) cultivation generally enhance nutrient accumulation mainly through increasing concentrations of NO3−, K+, Ca2+ and SO4− in the root zone. Among organic substrates, PV showed higher Ca but lower P concentration in tomato roots, when compared to CC. The growth of thin roots (0–0.2 mm diameter) was more vigorous under PV than under CC. The iTRAQ analysis identified several proteins related to nutrient accumulation in the root zone of organic substrates, such as calmodulinlike protein, high-affinity nitrate transporter 3.2-like protein and other mineral ion binding and transport proteins. Further studies need to be conducted to investigate how these proteins regulate nutrient accumulation under organic substrate cultivation. Author contributions JYX and JX performed the experiments and analyzed the data. JYX drafted the manuscript. WL developed the experimental design and revised the manuscript together with NG. Conflicts of interest The authors declare no conflict of interest. Acknowledgments This project was supported by Fruit Vegetables Innovation Team in 198
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Table 6 Differential abundance protein species (DAPS) identified in tomato roots related to nutrient accumulation stress response under water culture (WC), peat-vermiculite (PV) and coir (CC) cultivations. Accession
Protein species name
Mineral Ion Binding and Transport Related Proteins K4B6T5 Calmodulin-like protein 1 K4C1P5 Putative ATP synthase subunit delta', mitochondrial-like K4C1P6 Putative ATP synthase subunit delta', mitochondrial-like K4BIU4 Putative ATP synthase subunit delta', mitochondrial-like K4CBV3 Putative ABC transporter E family member 2-like Q4W5U8 FtsH protease K4BK64 High-affinity nitrate transporter 3.2-like K4C977 Hyoscyamine 6-dioxygenase K4CBX5 purple acid phosphatase polyphenol oxidase F K4DA09 Putative copper ion binding-like K4CN52 Putative ADP-ribosylation factor GTPase-activating protein AGD9-like Stress and defense Q05539 K4CQB7 K4CWC6 P15003 K4AX33 K4CPH4 K4BPW4 K4CVX0 K4CVX3 K4CVX7 K4CWC5 K4BVR5 K4BU44 K4BAN0 K4CWC6 K4ASJ5 K4BTH6 K4DH34 K4DHM7 K4CMC4 K4CX43 K4BAE6 K4BG30 K4BAL6 K4D1W1 K4C0T3
Acidic 26 kDa endochitinase Putative L-ascorbate peroxidase 2, cytosolic-like Pathogenesis-related protein STH-2 Suberization-associated anionic peroxidase 1 Peroxidase Disease resistance response protein 206 Putative universal stress protein A-like PIN-I protein Type I serine protease inhibitor Proteinase inhibitor I PR10 protein Peroxidase Protein CutA Ascorbate peroxidase Pathogenesis-related protein STH-2 Peroxidase Peroxidase Catalase Apyrase Peroxidase Putative plasma membrane-associated cation-binding protein 1-like Catalase Peroxidase Peroxidase Peroxidase Peroxidase
Beijing (BAIC01-2018) and Youth Research Fund of Beijing Academy of agricultural and Forestry Sciences (QNJJ201825).
CC vs. WC
CC vs. PV
WC vs. PV
1.812 0.814 1.182 1.034 1.008 0.879 0.541 1.721
0.942 0.498 0.925 0.521 0.550 0.438 0.965 0.599
0.453 0.531 0.573 0.504 0.541 0.498 1.784 0.348
1.735 1.820 1.613
0.763 0.684 0.871
0.440 0.376 0.540
1.861 1.779 1.752 1.569 1.549 0.647 1.885 2.294 1.388 2.386 1.454 0.612 1.484 1.462 1.752 2.055 1.616 1.995 2.735 0.490 1.942 0.794 1.817 1.781 0.574 0.440
0.953 1.131 0.839 0.854 0.883 1.010 3.079 0.943 0.401 0.525 0.841 0.344 1.232 0.931 0.839 0.615 0.627 1.043 0.697 1.015 1.159 1.198 0.687 0.819 1.081 1.921
0.512 0.636 0.479 0.544 0.570 1.560 1.633 0.411 0.289 0.220 1.729 1.777 0.665 0.637 0.479 0.299 0.388 0.523 0.255 2.072 0.597 1.509 0.378 0.460 1.884 2.094
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Influence of organic substrates on nutrient accumulation and proteome changes in tomato-roots Jiayi Xinga,b, Nazim Grudac, Jing Xionga, Wei Liua,b,
T
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a
Vegetable Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing, 100097, China Key Laboratory of Urban Agriculture (North China) by Ministry of Agriculture and Rural Affairs, Beijing, 100097, China c INRES – Horticultural Science, University of Bonn, 53121, Bonn, Germany b
A R T I C LE I N FO
A B S T R A C T
Keywords: Organic growing media Soilless culture Coir Peat-vermiculite substrate Nutrient accumulation Proteome
The gradually accumulation of nutrients in organic substrates during growing season may have adverse effects on plant development. This study was conducted to determine the mode of nutrient accumulation and alteration at molecular level induced by the excess nutrient stress in tomato. The ICP-MS was used to analyze mineral ions in root zone solution and nutrients uptake by tomato plants; and iTRAQ-based proteomic analysis was conducted to identify proteome changes in tomato roots. Coir and peat-vermiculite were used in substrate treated experiments. Results showed that the content of K+ was higher in coir than in peat-vermiculite whereas it was the opposite for Ca2+. The concentrations of NO3−, SO42- and Mg2+ were significantly higher in peat-vermiculite and coir substrates than in water culture. The peat-vermiculite substrates generally enhanced Ca uptake but reduced P uptake by plants, when compared to both coir and water culture. Compared to water culture, functional annotation analysis of the root proteome revealed that the excess nutrient accumulation induced complex proteomic alterations involved in mineral ion binding and transport. A total of 358 differentially abundant proteins (DAPS) were identified, including 11 mineral ion binding and transport related proteins, such as calmodulin-like protein and nitrate transporter 3.2 under peat-vermiculite and coir cultivations. RT-qPCR was used to validate nine genes encoding DAPS. We believe that these indicators will contribute to a better control of soilless culture systems and a waste reduction in production of tomatoes.
1. Introduction Organic substrates, such as peat and coir, are widely used in vegetable crop production around the world (Gruda, 2012; Savvas and Gruda, 2018). The physical properties of organic substrates depend on the materials and containers used, growing media (GM) compression or density, i.e. the amount of substrate in a certain volume, the applied irrigation system and root development (Gruda and Schnitzler, 2004). According to Gruda and Schnitzler (2004), under certain conditions, organic GM may have a limited air and low water buffer capacity, sometimes accompanied with failures and a bottleneck situation of nutrients (Gruda and Schnitzler, 1999). During the cultivation period, since nutrient supply and crop fertilizer absorption are not completely consistent, some mineral elements may gradually accumulate in the organic substrates around the root zone (Savvas and Gizas, 2002). Nutrient accumulation in the root zone can negatively influence crop growth through inducing osmotic stress in the rhizosphere and then limiting the absorption of water and nutrients by roots (Gruda, 2009).
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Moreover, it may lead to nutrient imbalance and ion toxicity in crops (Carmona et al., 2012; Urrestarazu et al., 2008), further resulting in yield decline and quality deterioration (Gruda and Schnitzler, 2004; Changa and Lee, 2016). In recent years, proteomics has been widely used to identify salt tolerance proteins in cucumber and other vegetable crops (Fan et al., 2015; Li et al., 2015; Jiang et al., 2017). Isobaric tags for relative and absolute quantitation (iTRAQ), a mass spectrometry based quantitative method, which can identify and quantify proteins of multiple high coverage samples at the same time (Wiese et al., 2007). To date, this method has been successfully applied in many studies related to a variety of abiotic stress conditions, because of its high sensitivity and more accurate quantification (Chen et al., 2016; Liu et al., 2017; Jiang et al., 2017). Changes in root proteomes is one of the most direct signals and metabolic factors that can be easily induced by suboptimal conditions in the root zone (Zhang et al., 2012). Therefore, investigating the protein changes in response to different cultivation substrates may help to reveal the potential mechanism of nutrient accumulation in the root
Corresponding author at: Vegetable Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing, 100097, China. E-mail address: [email protected] (W. Liu).
https://doi.org/10.1016/j.scienta.2019.03.054 Received 14 September 2018; Received in revised form 12 February 2019; Accepted 23 March 2019 0304-4238/ © 2019 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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zone (Asaduzzaman et al., 2013). Tomato is the most cultivated crop worldwide. The soilless culture system for growing tomatoes utilizes organic materials (e.g. peat and coir) as GM rather than rockwool. Nutrient accumulation in the root zone is a major limiting factor that could adversely affect plant growth (Xiong et al., 2017). Although previous studies have investigated the influences of different organic substrates on the retention and availability of nutrients in root zone, it is still unclear how organic substrates affect root protein changes under nutrient accumulation conditions (Sun et al., 2016). To meet this need, we investigated the characteristics of nutrient accumulation in tomato root zone under coir cultivation (CC) and peat-vermiculite cultivation (PV), while using water culture (WC) as control. Moreover, we used iTRAQ analysis to comprehensively assess dynamic changes of proteome in tomato roots under different cultivation systems (i.e. CC, PV and WC). The aims of this study were to investigate the expression and regulation mechanism of main ion channel proteins during nutrient accumulation process and to understand how tomato roots respond at protein levels to nutrient accumulation in organic GM.
Table 2 Nutrient formula of nutrient solution. Macroelement
Concentration (mmol L−1)
Microelement
Concentration (μmol L−1)
NO3− NH4+ H2PO4− K+ Ca2+ Mg2+ SO42−
15.4 1.4 1.8 9.3 3.9 1.4 2.1
Fe Mn Cu Zn B Mo –
14.7 27.8 0.8 6.7 4.2 0.07 –
2.3. Nutrient solution management A closed-loop soilless culture system was used. Each gutter was fed from a nutrient solution tank. The nutrient solution drained directly back into the same tank where it was mixed with the new solution. The compositions of standard nutrient solution were shown in Table 2. The electrical conductivity (EC) and pH of the nutrient solution tanks were monitored every week. To maintain the EC of 2.3 dS m−1, the nutrient tank was maintained at 200 L by replenishing with fresh solution containing fresh water, (EC 0.12 dS m−1, pH 7.18; Na+ 0.6 mmol L−1, Ca2+ 0.1 mmol L−1, Mg2+ 0.05 mmol L−1, SO42− 0.2 mmol L−1, NO3− 0.7 mmol L−1, NH4+ 0.05 mmol L−1and H2PO4− 0.02 mmol L−1). The nutrient solution was applied through a drip (average flow rate of 1.5 L h−1) irrigation system with one dripper per plant. The irrigation frequency and volume were the same for all substrate culture gutters. Nutrient solution was supplied four times per day (9:00, 11:00, 13:00, and 15:00) for 10 min each, irrigation volume was 2 L per plant. For all water culture gutters, the nutrient solution was circulated for 30 min. in every two hours from 8:00 am to 6:00 pm. Every 2 months, the nutrient solution tank was washed and the nutrient solution in the tank was completely refreshed.
2. Materials and methods 2.1. Experimental site and crop planting The experiment was conducted in a climate-controlled greenhouse in Beijing Vegetable Research Center, Beijing Academy of Agriculture and Forestry Sciences in Beijing. The average light intensity ranged from 18.3–136.8 μmol m−2 s-1, and the average temperature was at 14.0–23.0℃, respectively. Tomato (Solanum lycopersicum) ‘Lucius F1’ seeds were sown in plug tray and thirty-three days later they were transplanted into coir cubes (10 cm × 10 cm). Thirty-three days after planting in the substrate cube, the tomato seedlings were transplanted into substrate slabs (CC and PV) or nutrient solution (WC). The planting density was 2.4 crops m−2.
2.4. Root-zone solution analysis From 3 weeks after transplanting, root-zone solution was sampled every two weeks. Root-zone solution (100 ml) was collected with a root solution extractor installed between the crops. The samples were stored at 2 °C until further analyzing. The EC was measured using a multi meter (Multi 3420 SET C., WTW, Germany). K+, Ca2+, Mg2+, NO3−and H2PO4− were assayed by inductively coupled plasma spectrometry (ICPE-9000, Shimazu, Japan).
2.2. Experimental design Coir was bought from Jiffy Group in Netherland. Both peat and vermiculite were bought from Beijing Lide Agricultural S&T Development Company in China. The mixture of peat and vermiculite (v/v, 2:1) (PV) were used as cultivation substrates in the experiment. Selected characteristics of different substrates were showed in Table 1. The experiment was a completely randomized block design with three replicates and each replicate contained a cultivation gutter of 10 m long. Tomato crops were planted with 30-cm plant spacing and each gutter had 30 plants. For each substrate culture gutter, 10 substrate slabs (100 cm × 20 cm × 7.5 cm) were installed. For each water culture gutter, semicircular PVC pipe of 32 cm in diameter was used. The depth of the nutrient solution was 10 cm.
2.5. Analysis of root system development 50, 80 and 107 days after transplanting, roots of one representative plant from replicate of all the treatment conditions were sampled. Roots were washed on 0.5 mm diameter sieve, scanned (SNAPSCAN 1236, AGFA, Germany) and then analyzed with the WinRHIZO software package (Regent Instruments Inc. Canada) for root length, diameter and surface area. The roots were further divided into four classes according to their diameters (i.e. 0–0.2, 0.2−0.6, 0.6−0.9, > 0.9 mm).
Table 1 Selected physical and chemical properties of coir and peat-vermiculite. Properties
Coir
Peat-vermiculite
EC (dS m−1) pH C (%) N (mg kg−1) P (mg kg−1) K (mg kg−1) Ca (mg kg−1) Mg (mg kg−1) S (mg kg−1) Total Porosity (%) Bulk density (g cm−3)
0.10 6.1 49.5 44 38 1560 58 55 405 85.6 0.2
1.10 7.1 15.9 64 42 246 1668 636 645 66.0 0.4
2.6. Plant nutrient analysis Similarly to roots analyses, roots, stems, leaves and fruits of one plant from each replicate of all treatment conditions were sampled on days 50, 80 and 107 after transplanting. After washing with distilled water, they were dried in a ventilated oven at 75 °C to constant weight. Nutrient contents in roots, stems, leaves and fruits samples were analyzed. The contents of K, Ca, Mg and P were assayed after digestion with H2SO4-HNO3-HClO4 (H2SO4: HNO3: HClO4 = 1 ml: 5 ml: 1 ml) by inductively coupled plasma spectrometry (ICPE-9000, Shimazu, Japan). 193
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2.7. Protein isolation, digestion, and labeling with iTRAQ reagents On day 107 after transplanting, the whole roots of one plant from each replicate were harvested. Protein extraction of roots was conducted using the phenol extraction/methanol-ammonium acetate precipitation approach (Faurobert et al., 2007). Protein concentration was determined by BCA Protein Quantitation Kit (Sangon Biotech, Shanghai, China). After reduction and cysteine-blocking as described in the iTRAQ protocol (AB Sciex, Concord, ON), solutions containing 100 μg protein were digested overnight at 37 °C with sequence grade modified trypsin (Promega, Madison, WI) and then labeled with different iTRAQ tags as follows: IT113 and IT114 for WC samples; IT117 and IT118 for PV samples; IT119 and IT121 for CC samples. The labeled samples were then pooled and dried on a rotary vacuum concentrator.
Fig. 1. Electrical conductivity (EC) in root-zone solution under water culture (WC), peat-vermiculite (PV) and coir (CC) cultivations. The vertical bars represent the standard errors. Black letter, green letter and purple letter WC, PV and CC cultivations, respectively (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
2.8. LC–MS/MS analysis The peptide mixtures were pressure-loaded onto a fused silica capillary column packed with 3-μm dionex C18 material (RP; Phenomenex). The RP sections with 100 Å were 15 cm long, and the column was washed with buffer A (water, 0.1% formic acid) and then buffer B (ACN (Acetonitrile), 0.1% formic acid). After desalting, a 5mm, 300-μm C18 capture tip was placed in line with an Agilent 1100 quaternary HPLC (High Performance Liquid Chromatography) and analyzed using a 12-step separation.
3. Results 3.1. EC in root-zone solution The EC in root zone solution of all substrates was maintained at relatively stable levels in the first 9 weeks after transplanting, and then increased gradually during the next 6 weeks (Fig. 1). In the 15th week after transplanting, the EC in root-zone solution reached 7.71 dS m−1 in CC, 5.86 dS m−1 in PV, and 4.21 dS m−1 in WC, inferring the serious nutrient accumulation situation in root zone under substrate cultivation systems.
2.9. DAPS data analysis Tandem mass spectra were searched against mascot 2.1 (Local Host) uniprot_solanum_lycopersicum. Fasta. The search results were then filtered using a cutoff of 1% for peptide false identification rate. Peptides with Z score < 4 or Delta-Mass > 5 ppm were rejected. Furthermore, the minimum number of peptides to identify a protein was set to 1. The default parameters for the Quantitative software Profile Analysis 2.0 were used throughout the analysis. Bioinformatics analysis was carried out to categorize proteins based on biological processes, cellular component and molecular function using annotations in Protein Analysis Through Evolutionary Relationships (PANTHER) database v 6.1(www. pantherdb.org), which is in compliance with gene ontology (GO) standards.
3.2. Root length, root surface area and average root diameter for tomato A smaller root surface area was found in WC, when compared to PV and CC (Table 3). On days 80 and 107 after transplanting, both the root length and surface area were larger in WC than in PV and CC. With respect to average root diameter, it was significantly higher in CC than in PV on day 50, and in WC and PV on days 80 and 107 after transplanting. Overall, thin roots (0-0.2 mm diameter) had the highest proportion of total root length under all treatments (Fig. 2). On days 80 and 107 after transplanting, the proportion of thin roots was significantly higher in WC and PV than in CC. However, reverse trends were found in relatively thicker roots (i.e. 0.2−0.6 mm, 0.6−0.9 mm and > 0.9 mm diameter) among treatments. The highest proportion of total surface was generally found in thin roots (0-0.2 mm diameter) under WC and PV, but in relatively thicker roots (> 0.2 mm) under CC (Fig. 3).
2.10. RNA extraction and RT-qPCR 107 days after transplanting, total RNA extraction and reversetranscription quantitative PCR (RT-qPCR) analysis of roots were carried out according to the method as described in Sun (2016). All the primer pairs used for RT-qPCR were designed using the Beacon Designer 7.0 and provided in Table S1. The data were processed with the 2−△△CT method as described by Livak and Schmittgen (2001).
3.3. Ions accumulation in root zone solution The concentration of K+ in root-zone solution was significantly higher in CC than in WC and PV, and in WC than in PV (Table 4). The concentration of Ca2+ in root-zone solution was higher in PV than in CC. With respect to Mg2+, NO3− and Ca2+, the ion concentrations were higher in PV and CC than in WC. Moreover, the concentration of H2PO4− in root-zone solution was significantly higher in CC than in WC and PV.
2.11. Fruit yield During fruit ripening period, for each cultivation gutter, fruits were harvested from 18 plants from each replicate to measure fresh yield. At the end of the cropping season, the fresh yield of each harvest was summed up as the total yield (t hm−2).
3.4. Nutrient distribution in tomato plants and fruit yield 2.12. Statistical analysis As shown in Table 5, the investigated systems generally had different effects on the nutrient distribution among different plant organs. In roots, only the concentration of Ca was significantly influenced and increased by PV compared to WC and CC. In both stem and leaf, the concentration of P was significantly lower in PV than in WC and CC, but
Statistical analysis was carried out with SPSS 20.0 software (SPSS statistical package, Chicago, IL, USA). Multiple comparisons using Duncan’s test were done whenever the ANOVA indicated significant differences (P ≤ 0.05). 194
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Table 3 The dynamics of root length, root surface area and average root diameter for tomato under water culture (WC), peat-vermiculite (PV) and coir (CC) cultivations. Treatment
WC PV CC a
Root surface area (m2)
Root length (m)
Average root diameter (mm)
50 d
80d
107 d
50 d
80d
107 d
50 d
80d
107 d
338.5 a 434.7 a 399.2 a
1361.2 a 901.2 b 757.2 b
1556.2a 1251.9ab 839.3 b
0.25 b 0.37 a 0.35 a
1.14 a 0.79 b 0.73 b
1.10 a 0.89ab 0.73 b
0.24ab 0.22 b 0.28 a
0.22 b 0.24 a 0.31 a
0.23 b 0.21 b 0.28 a
The same letter denotes no significant difference among different treatments (LSD test at P < 0.05).
accumulation stress reaction. Moreover, some DAPS were found to be remarkably enriched in stress-response-related biological processes, such as “response to oxidative stress” and “response to biotic stimulus”. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis (p < 0.05), the DAPS were mainly enriched into plant biology processes, including ribosome, phenylpropanoid biosynthesis, carbon metabolism, chemical carcinogenesis, protein processing in endoplasmic reticulum, drug metabolism - cytochrome P450, metabolism of xenobiotics by cytochrome P450, starch and sucrose metabolism and glutathione metabolism (Fig. S2).
a reverse trend was found in the concentration of ca. Additionally, the concentrations of Mg in both stem and leaf were significantly higher in PV and CC than in WC. No significant difference in nutrient concentration was generally found in the fruit among treatments. For fruit yields only the first two trusses were recorded and no significant differences were found between the treatments. 3.5. Identification of DAPs using iTRAQ LC–MS/MS A total of 21,284 peptides and 4163 non-redundant protein groups were identified in tomato roots after 15 weeks of transplanting, and the labeling efficiency was 98.24%. To obtain differentially abundant proteins (DAPs), proteins were compared between each pair of treatments (WC, CC and PV). The proteins with ratio values of more than 1.2 or less than 0.83 (P < 0.05) were defined as DAPs. A total of 358 DAPs in the above 3 comparison schemes were identified in all 3 replicates. Based on this criteria, 128, 51, and 154 proteins were identified to be up-accumulated, and 83, 32, and 204 proteins were down-accumulated during CC vs. WC, CC vs. PV, and WC vs. PV, respectively (Fig. 4). GO annotations (GO) indicated that the 358 identified proteins were involved in 30 biological processes, 25 cellular components and 30 molecular functions (Figs. 5 and S1). Among these categories, “oxidation-reduction process”, “metabolic process”, “response to oxidative process”, “hydrogen peroxide catabolic process” and “carbohydrate metabolic process” were the most common biological processes. In cellular component category, “extracellular region”, “integral component of membrane” and “membrane” were the central categories. “The metal ion binding”, “oxidoreductase activity” and “hydrolase activity” were the most dominant categories in molecular function. Several important cellular components were closely related to the transport activities of water and various ions such as “plasma membrane” and “proton-transporting ATP synthase complex, catalytic core F(1)”. Further analysis identified a few of DAPS that were closely correlated with “nitrate transport,” “metal ion binding” and “zinc ion binding”, suggesting that those ion channels might play an important role in nutrient
3.6. Validation of DAPs by RT-qPCR The expression of nine genes, which were associated with ion absorption and transport-related proteins (SlCML1, SlNTR3.2, SlMscL10, SlPCaP1 and SlTMP1), carbohydrate metabolism (SlCA3), auxin-activated signaling pathway signaling pathway (SlARF18), response to oxidative stress (SlCAT3) and stress and defense (SlPHOS32), were determined by RT-qPCR to confirm the relationships between the expression changes of RNA and corresponding proteins (Fig. 6; Table 4). A majority of the nine genes showed a matching abundance level from mRNA to protein when the plants were treated under different substrates. However, the genes encoding carbonic anhydrase3 (SlCA3) and plasma membrane-associated cation-binding protein 1 (SlPCaP1) were inversely correlated between the mRNA and protein changes. 4. Discussion Currently, the soilless culture systems are regarded as a major technological component of modern greenhouses (Grunert et al., 2016). The independence from the soil as a rooting medium enables optimization of both physical and chemical characteristics in the root environment and a more efficient control of pathogens without the need to apply soil fumigation (Gruda, 2012). Both water culture systems with merely nutrient solution as root environment and cultivation on porous
Fig. 2. Ratio of root length with certain diameter to total root length for tomato under water culture (WC), peat-vermiculite (PV) and coir (CC) cultivations. The vertical bars represent the standard errors. Different letters indicate significant difference between treatments according LSD test at P < 0.05. 195
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Fig. 3. Ratio of root surface area with certain diameter to total root surface area for tomato under water culture (WC), peat-vermiculite (PV) and coir (CC) cultivations. The vertical bars represent the standard errors. Different letters indicate significant difference between treatments according LSD test at P < 0.05.
growing media which create a matrix that can retain both air and water at suitable ratios for plant growth included in these systems (Savvas and Gruda, 2018). In this study we used a peat-vermiculite, a coir and water culture system, in order to investigate the expression and regulation mechanism of main ion channel proteins and the response of tomato roots at protein levels to nutrient accumulation in soilless culture systems. The root is the main organ of a plant to absorb water and nutrient. The root epidermis is the region with the highest absorption activity, and the increase of root length could improve the efficiency of water and nutrient use (Rich et al., 2011). Therefore, the root surface area and root length are the key indicators to measure root absorption capacity. In this study, WC provided a relatively normal environment for root growth compared to substrate cultures according to the root zone EC (Fig. 1). The EC in root zone solution of substrate treatments was maintained at relatively stable levels during the first 9 weeks after transplanting and increased much faster than WC thereafter. Correspondingly, there was no significant difference in root length among treatments 50 days after transplanting, while both root length and root surface area of PV and CC were significantly lower than those of WC 80 days after transplanting (Table 3). It can be postulated that as plants are exploring for nutrients, a larger root system should be developed. Gruda reported a higher root development with longer roots in wood fiber substrates with a lower level of nitrogen supply as in good balanced nitrogen treatment (Gruda et al., 2012). CC had an even higher root zone EC than that of PV, where root length and root surface area of CC were also lower than that of PV. The results indicated that nutrient accumulation in substrate affected the root growth, especially for thin roots (0–0.2 mm diameter) which had the largest proportion of total length and surface area (Figs. 2 and 3). From the beginning, average root diameter of CC was higher than that of PV and WC. During the whole study period, the average root diameter of each treatment kept the same. These results suggested that the average root diameter was mainly affected by the physical properties of substrates (Kato et al., 2010). Nutrient accumulation in root zone is critical for plant growth under substrate cultivation (Changa and Lee, 2016). For all substrates, most
Table 5 Nutrient distribution in tomato plants, cultivated in water culture (WC), peatvermiculite (PV) and coir (CC) cultivations. Organs
Root
Stem
Leaf
Fruit
Treatment
WC PV CC WC PV CC WC PV CC WC PV CC
Nutrient (mg/g dry matter) P
K
Ca
Mg
2.2 a 2.2 a 2.4 a 9.4 a 6.1 b 9.5 a 11.5 a 6.3 b 10.9 a 6.1 a 5.3 a 6.2 a
3.3 a 3.3 a 4.1 a 127.4 128.5 144.6 143.3 120.8 127.9 106.5 110.0 119.1
32.4 b 37.1 a 34.1 b 25.7 b 31.5 a 25.9 b 83.5 b 91.4 a 79.3 b 1.1 a 1.8 a 2.0 a
3.7 a 3.2 a 4.0 a 5.2 b 7.5 a 7.5 a 10.5 b 13.4 a 14.7 a 3.5 a 3.9 a 3.9 a
a a a a a a a a a
All data were average value of seven sampling times. a The same letter denotes no significant difference among different treatments (LSD test at P < 0.05).
mineral ions increase gradually as the growing time extended, resulting in gradually increased EC in root zone (Xiong et al., 2017). Organic substrates release certain kinds of ions to solution meanwhile absorb other ions. In this study, we noted that in the root zone of plants cultivated in coir a relatively high K+ content were accumulated, while peat-vermiculite had a high Ca2+ content. The concentration of K+ in root zone solution was significantly higher in CC than in PV and WC, and Ca2+ content was higher in PV than in CC and WC (Table 1, Table 3), probably due to that CC released K+ while PV adsorbed K+ to and from solution, respectively (Barrett et al., 2016; Rippy and Nelson, 2007). Compared with WC, the concentrations of NO3−, SO4− and Mg2+ were significantly higher in the root zone of organic substrates CC and PVC (Table 4), and this might be an important factor leading to the nutrient accumulation stress (Fig. 1). The concentration of Ca2+ in root zone solution was higher in PV than in CC and WC. The Ca2+ concentration in root, stem and leaf of PV was also significantly higher
Table 4 Some nutrient in root zone solution under water culture (WC), peat-vermiculite (PV) and coir (CC) cultivations. Treatment
K+ mmol L−1
Ca2+ mmol L−1
Mg2+ mmol L−1
NO3− mmol L−1
SO4− mmol L−1
H2PO4− mmol L−1
WC PV CC
11.43 b 7.45 c 18.07 a
5.62 ab 9.03 a 4.35 b
2.18 b 3.77 a 3.66 a
12.65 b 54.32 a 59.61 a
1.95 b 7.58 a 8.57 a
1.95 b 2.58 b 5.57 a
All data are average value of seven sampling times. a The same letter denotes no significant difference among different treatments (LSD test at P < 0.05). 196
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used to understand the molecular response mechanisms of tomato root under nutrient accumulation stress, and a total of 358 were DAPs identified. Among them, a number of proteins that regulate the binding and transport of ions were identified with altered abundance under treatment conditions (Table 6). Calcium signaling pathways play an important role in triggering complex stress responses in plants (Grant et al., 2015). Calmodulin (CaM) and calmodulin-like proteins (CMLs) are major Ca2+ sensors, playing critical roles in interpreting encrypted Ca2+ signals under stress.(Wang et al., 2015) AtCML8 and CML37 were found to be induced by salt treatment (Park et al., 2010; Vanderbeld and Snedden, 2007). CML1 is an important Ca2+ signaling sensor, and it has been shown to be involved in many responses to salt stress (Srivastava et al., 2013). This protein was significantly up-accumulated in CC and PV compared to WC, indicating that calcium signaling pathway plays an essential and positive role in tomato response to nutrient accumulation stress under organic substrate cultivation. K+ is an essential ion for many physiological processes, especially for enzyme activation and protein synthesis under stress (Anschütz et al., 2014). In our data, five H+-ATPase activity related protein, including three F0F1type H+-ATP synthase delta subunits, FtsH protease and ABC transporter E family member 2-like, were up-accumulated in PV, compared to WC and CC. F0F1-type H+-ATP could affect the transport of K+ in cotton under salt stress (Gong et al., 2017), and we hypothesized that it may be a key factor leading to the lower concentration of K+ in PV, in addition to a part of K+ absorbed by PV (Xiong et al., 2017). The concentration of NO3− in root-zone solution was the highest of all ions, and it is also the largest contribution to nutrient accumulation stress. Nitrate transporters (NTR) are induced by nitrate, and can make plants grow normally under various nitrogen concentrations (Kiba et al., 2012). High-affinity nitrate transporter 3.2-like protein had a highest expression in WC, and this may explained why the concentration of NO3- was the lowest in WC. It shows that increasing the expression of high-affinity nitrate transporter 3.2-like protein can enhance plant resistance nutrient accumulation stress. We identified several additional proteins related to mineral ion binding, including hyoscyamine 6-dioxygenase, purple acid phosphatase (PAPs), polyphenol oxidase (PPO) F, putative copper ion bindinglike and putative ADP-ribosylation factor GTPase-activating protein AGD9-like. PGPAP18 may play a defensive role against environmental
Fig. 4. Identification and statistics of differential abundance protein species (DAPS) under water culture (WC), peat-vermiculite (PV) and coir (CC) cultivations. (A) Number of up- or down-accumulated protein species between two different cultivations. (B) Venn diagram analysis of up-accumulated and downaccumulated protein species.
compared to WC and CC. Accumulation of mineral ions could lead to an increase in EC value, resulting in nutrient accumulation stress in root zone of organic substrate crops (Barrett et al., 2016). An iTRAQ-based quantitative was
Fig. 5. The most significantly-enriched gene ontology (GO) terms of differential abundance protein species (DAPS) under water culture (WC), peat-vermiculite (PC) and coir (CC) cultivations. 197
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Fig. 6. Relative mRNA expression analysis using RT-qPCR on nine protein species under water culture (WC), peat-vermiculite (PV) and coir (CC) cultivations. The expression level under WC was set to a value of 1. Each bar shows the mean ± SE (n = 3). Letters above the columns indicate significant differences at p < 0.05 according to Duncan’s multiple range test. Nine protein species were auxin response factor 18-like (ARF18), carbonic anhydrase3 (CA3), calmodulin-like protein 1 (CML1), mechanosensitive ion channel protein 10 (MscL10), high-affinity nitrate transporter 3.2-like (NTR3.2), plasma membrane-associated cation-binding protein 1-like (PCaP1), suberization-associated anionic peroxidase 1-like (TMP1), universal stress protein PHOS32 and Catalase-3 (CAT 3).
5. Conclusion
stress and endow stress-tolerant plant with stress adaptation. (Reddy et al., 2017). PPOs are Zn- and Cu-responsive and involved in metal ionassociated gene networks in Populus (Lu et al., 2011), and overexpression of a potato PPO cDNA could improve the disease resistance in tomato (Li et al., 2002). These proteins were all up-accumulated in CC and PV, compared to WC, implying that mineral ion binding pathway might be managed by complex regulatory mechanism to cope with nutrient accumulation stress in tomato. The generation of ROS is one of the main stress-induced universal consequences in plants (Meloni et al., 2003; Guo et al., 2012). It can not only cause irreversible damage to cells, but also attack macromolecules. A series of antioxidants and enzymes are needed to scavenge ROS in plants (Sun et al., 2017). In this study, we identified a total of 26 DAPs related to oxidative stress response (Table 6). Among those DAPs, peroxidase and catalase were two major categories, most of which were up-accumulated in organic substrates (CC and PV) compared to water culture (WC), suggesting that their significant roles in response to nutrient accumulation stress. Additionally, a putative universal stress protein A-like, another protein species related to stress tolerance, was also identified (Table 6). This protein has been demonstrated with functions in enhancing plant’s resistance to unfavorable conditions through forming a natural biological defense mechanism (Tkaczuk et al., 2013). The expression of putative universal stress protein A-like was markedly higher in CC than in PV and WC, indicating that tomato plants expressed a more active response to the stress conditions on the CC substrate.
Compared with water culture, organic substrates (PV and CC) cultivation generally enhance nutrient accumulation mainly through increasing concentrations of NO3−, K+, Ca2+ and SO4− in the root zone. Among organic substrates, PV showed higher Ca but lower P concentration in tomato roots, when compared to CC. The growth of thin roots (0–0.2 mm diameter) was more vigorous under PV than under CC. The iTRAQ analysis identified several proteins related to nutrient accumulation in the root zone of organic substrates, such as calmodulinlike protein, high-affinity nitrate transporter 3.2-like protein and other mineral ion binding and transport proteins. Further studies need to be conducted to investigate how these proteins regulate nutrient accumulation under organic substrate cultivation. Author contributions JYX and JX performed the experiments and analyzed the data. JYX drafted the manuscript. WL developed the experimental design and revised the manuscript together with NG. Conflicts of interest The authors declare no conflict of interest. Acknowledgments This project was supported by Fruit Vegetables Innovation Team in 198
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Table 6 Differential abundance protein species (DAPS) identified in tomato roots related to nutrient accumulation stress response under water culture (WC), peat-vermiculite (PV) and coir (CC) cultivations. Accession
Protein species name
Mineral Ion Binding and Transport Related Proteins K4B6T5 Calmodulin-like protein 1 K4C1P5 Putative ATP synthase subunit delta', mitochondrial-like K4C1P6 Putative ATP synthase subunit delta', mitochondrial-like K4BIU4 Putative ATP synthase subunit delta', mitochondrial-like K4CBV3 Putative ABC transporter E family member 2-like Q4W5U8 FtsH protease K4BK64 High-affinity nitrate transporter 3.2-like K4C977 Hyoscyamine 6-dioxygenase K4CBX5 purple acid phosphatase polyphenol oxidase F K4DA09 Putative copper ion binding-like K4CN52 Putative ADP-ribosylation factor GTPase-activating protein AGD9-like Stress and defense Q05539 K4CQB7 K4CWC6 P15003 K4AX33 K4CPH4 K4BPW4 K4CVX0 K4CVX3 K4CVX7 K4CWC5 K4BVR5 K4BU44 K4BAN0 K4CWC6 K4ASJ5 K4BTH6 K4DH34 K4DHM7 K4CMC4 K4CX43 K4BAE6 K4BG30 K4BAL6 K4D1W1 K4C0T3
Acidic 26 kDa endochitinase Putative L-ascorbate peroxidase 2, cytosolic-like Pathogenesis-related protein STH-2 Suberization-associated anionic peroxidase 1 Peroxidase Disease resistance response protein 206 Putative universal stress protein A-like PIN-I protein Type I serine protease inhibitor Proteinase inhibitor I PR10 protein Peroxidase Protein CutA Ascorbate peroxidase Pathogenesis-related protein STH-2 Peroxidase Peroxidase Catalase Apyrase Peroxidase Putative plasma membrane-associated cation-binding protein 1-like Catalase Peroxidase Peroxidase Peroxidase Peroxidase
Beijing (BAIC01-2018) and Youth Research Fund of Beijing Academy of agricultural and Forestry Sciences (QNJJ201825).
CC vs. WC
CC vs. PV
WC vs. PV
1.812 0.814 1.182 1.034 1.008 0.879 0.541 1.721
0.942 0.498 0.925 0.521 0.550 0.438 0.965 0.599
0.453 0.531 0.573 0.504 0.541 0.498 1.784 0.348
1.735 1.820 1.613
0.763 0.684 0.871
0.440 0.376 0.540
1.861 1.779 1.752 1.569 1.549 0.647 1.885 2.294 1.388 2.386 1.454 0.612 1.484 1.462 1.752 2.055 1.616 1.995 2.735 0.490 1.942 0.794 1.817 1.781 0.574 0.440
0.953 1.131 0.839 0.854 0.883 1.010 3.079 0.943 0.401 0.525 0.841 0.344 1.232 0.931 0.839 0.615 0.627 1.043 0.697 1.015 1.159 1.198 0.687 0.819 1.081 1.921
0.512 0.636 0.479 0.544 0.570 1.560 1.633 0.411 0.289 0.220 1.729 1.777 0.665 0.637 0.479 0.299 0.388 0.523 0.255 2.072 0.597 1.509 0.378 0.460 1.884 2.094
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