Quantitative proteomics suggests changes in the carbohydrate metabolism of maize in response to larvae of the belowground herbivore Holotrichia parallela

Plant material and insect treatment

Maize plants were sown in plastic pots (height, 20 cm; diameter, 15 cm) containing a mixture of pasteurized field soil and sand (3:1), as previously described (Pan et al., 2020). The original maize seedlings were supplied by Professor Yaping Yuan from Jilin University. The soil was excavated from the field of Changchun in Jilin Province (latitude 50°54′19″N, longitude 125°15′45″E) and was sieved through a 2-mm mesh before use. The seedlings were grown in a climate chamber (22 ± 2 °C, 70% relative humidity, 16 h:8 h light:dark). Thirteen days later, plants with two expanded primary leaves were used for the experiments (Erb et al., 2009). H. parallela larvae were collected from a local maize field in Changchun, China, and were reared with maize seedling roots at room temperature until use. For the proteomics analysis of maize seedlings, plants were either infested with one third-instar H. parallela larvae or left herbivore-free. After 24 h of infestation, the herbivores were gently removed, and the plants were harvested. The timing of this step was chosen based on previous studies showing a strong induction of defenses after 24 h of infestation (Marti et al., 2013; Glauser et al., 2011). The roots and leaves from the harvested plants were carefully excised from the shoots and weighed immediately to determine their fresh weight. Then, the roots were wrapped in aluminum foil, immediately frozen in liquid nitrogen, and stored at −80 °C until use.

Protein extraction

Protein extraction was performed using a modified method (Isaacson et al., 2006). Briefly, the root samples from maize seedlings were frozen in liquid nitrogen and pulverized. The samples were mixed with five volumes of TCA/acetone (1:9) by vortexing and incubated at −20 °C for 4 h. The supernatant was discarded after centrifugation at 6000 g for 40 min. The precipitant was washed three times with pre-cooling acetone and was air-dried. The pellet was dissolved in SDT buffer containing 4% SDS, 150 mM Tris-HCl, 1mM DTT, (pH 8). The lysate was sonicated then boiled for 15 min. Finally, the supernatant was filtered through 0.22-µm filters after centrifugation at 14,000 g for 40 min. The supernatant was quantified with the BCA Protein Assay Kit (Bio-Rad, USA). The quality of the protein sample was measured by SDS-PAGE.

TMT labeling and HPLC fractionation

Two hundred micrograms of protein for each sample was incorporated in 30 µl SDT buffer. The detergent, DTT and other low-molecular-weight components were removed using UA buffer (8 M urea, 150 mM Tris-HCl at pH 8.0) by repeated ultrafiltration. Then, 100 µl iodoacetamide (100 mM IAA in UA buffer) was added to block reduced cysteine residues, and samples were incubated for 30 min in darkness. The filters were washed with 100 µl UA buffer three times and then washed with 100 µl 100 mM triethylammonium bicarbonate (TEAB) twice. Finally, the protein from each sample was digested with trypsin (Promega, USA) at a protein/trypsin ratio of 40:1 overnight at 37 °C. The peptides were labeled using TMT six-plex isobaric label reagent (Product No: MAN0011639, Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. The sample peptides were fractionated using alkaline reverse-phase HPLC with an Agilent 300 Extend C18 column. The wavelength used for the detection of peptides was 250 nm. Briefly, the peptides were first separated into 80 fractions over 80 min using a gradient of 2–60% acetonitrile in 10 mM ammonium bicarbonate (pH 10). Then, the peptides were combined into 18 fractions and dried by vacuum centrifugation.

LC-MS/MS analysis

Each fraction was dissolved in 0.1% formic acid and loaded onto an Acclaim PepMap 100 reversed-phase C18 trap column (Thermo Fisher, Shanghai, China). Separation of peptides was performed using an EASY-nLC system and then subjected to tandem mass spectrometry Q EXACTIVE (Thermo Fisher Scientific) for data-dependent acquisition (DDA) detection by nano-electrospray ionization. The main parameters for MS analysis were collision energy, 30 eV; precursor scan range, 300–1,800 m/z; dynamic exclusion duration, 40.0 s; automatic gain control for full MS target, 3e6.

Database search

The MS/MS raw data were searched using the Mascot search engine embedded into Proteome Discoverer 1.4. The main Mascot search parameters were as follows: carbamidomethyl (C), TMT6/10plex (N-term), TMT6/10plex (K) as fixed modifications, and oxidation (M) as variable modifications. Additionally, 20 ppm of the peptide mass tolerance and 0.1 Da of the fragment mass tolerance was allowed. To reduce the probability of false peptide identification, only peptides with significance scores at the 95% confidence interval with a Mascot probability identity threshold were considered to be identified. The global false discovery rate was <1%, and each confident protein identification involved at least one unique peptide. Proteins quantitatively detected in all six samples were used to further analyze the abundance change between the control and treatment. Differentially abundant proteins (DAPs) were identified based on the following criteria: P-values smaller than 0.05 and a mean relative abundance > 1.2 or <0.83. The mass spectrometry proteomics data have been deposited to the Proteome EXchange Consortium via the PRIDE partner repository with the dataset identifier PXD018939.

Bioinformatics and annotations

To determine the functional characterization of proteins, proteins and differentially abundant proteins (DAPs) were mapped with Gene Ontology (GO) annotation (http://www.geneontology.org). All proteins were grouped into three major categories: biological processes, cellular components and molecular functions. The Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.kegg.jp/kegg/pathway.html) was employed to predict each DAP’s main metabolic pathway classification. SubLoc (Guo et al., 2004) was applied to predict the subcellular localization of each identified protein.

Herbivory-induced reconfiguration of primary metabolism in roots

To evaluate whether belowground herbivory reconfigures the primary metabolism in roots, we analyzed the contents of soluble sugar. Sucrose and hexose contents were determined using a sucrose, fructose and glucose assay kit (Mlbio, Shanghai, China) following the manufacturer’s instructions. The concentrations of glucose, fructose and sucrose were calculated using the calibration curves generated from corresponding standard solutions.

Statistical analysis

The experiments were performed with three biological replicates, and plant materials from three seedlings were pooled for each biological replicate. The results are presented as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) with Tukey’s test was conducted on the data, and p-values < 0.05 were considered to be significant.

Global proteomic changes in maize after H. parallela larvae infestation

H. parallela larvae infestation significantly affected the fresh masses of maize roots and leaves, on average, 42.6% and 16.2% lighter compared to controls, respectively (Fig. 1). To investigate the changes in protein abundance in response to H. parallela larval attack in maize roots, comparative proteomics-based Tandem Mass Tag (TMT) labeling technology was conducted. After processing MS/MS spectra in Mascot software, a total of 57,568 spectra were generated from TMT analysis, yielding 17,349 matched peptides, 11,681 unique peptides, 4,530 matched proteins and 3,229 quantified proteins (Fig. 2A).

According to a criterion of 95% significance and a 1.2-fold cutoff, a total of 654 proteins were identified as DAPs between the attacked roots and the control roots. Among these DAPs, 351 and 303 were identified as upregulated and downregulated proteins, respectively, with fold changes of 0.44 to 2.63 and protein masses of 1.59 to 549.34 kDa (Fig. 2B). All detailed information is listed in Table S1.

Enrichment analysis of DAPs under H. parallela larval attack

The biological functions of the DAPs could also be identified by their GO annotations. DAPs were divided into three categories: cellular component, biological process and molecular function. For the ‘biological process’ category, the DAPs were enriched in several GO terms, such as ‘cellular process’, ‘metabolic process’, ‘organic substance metabolic process’, and ‘cellular metabolic process’ (Fig. 3A). In the ‘cellular component’ category, most of the enriched proteins were related to ‘cell’, ‘cell part’, and ‘intracellular’ terms (Fig. 3B). For the ‘molecular function’ category, the highly enriched proteins were associated with ‘nucleic acid binding’, ‘structural molecule activity’, and ‘RNA binding’ (Fig. 3C).

Proteins in the same pathway presumably perform their biological function collectively. Pathway enrichment analysis using the KEGG database was performed to characterize the biological function of H. parallela larvae-affected proteins. Among the 654 identified DAPs, 222 (33.94%) DAPs were assigned to 66 specific KEGG pathways. The majority of DAPs were associated with ribosome (32 DAPs), glycolysis/gluconeogenesis (11 DAPs), amino sugar and nucleotide sugar metabolism (8 DAPs), fructose and mannose metabolism (7 DAPs), pentose phosphate pathway (7 DAPs) and RNA degradation (7 DAPs) (Fig. 3D).

DAPs involved in carbohydrate and energy metabolism

Carbohydrate and energy metabolism are one of the most basic metabolic pathways, and its mainly physiological function is to provide energy and a carbon source for many reactions and processes inside cells. Twenty-three DAPs involved in carbohydrate and energy metabolism were identified in maize roots responding to H. parallela larval attack, including proteins associated with glycolysis, gluconeogenesis, pentose phosphate pathway, and fructose and mannose metabolism (Table 1). The subcellular localization of most identified proteins and the other proteins related to carbohydrate and energy metabolism was cytoplasmic and mitochondrial, except for B6TT00 in the extracellular space (Table 1). Among these DAPs, 15 proteins were upregulated subsequent to H. parallela larvae in maize, with a ratio ranging from 1.214 to 1.679, including three dehydrogenases, two epimerases, two aldolases, two kinases and one protein from each of the isomerase, uridylyltransferase, diphosphorylase, mutase, galactosidase and uncharacterized protein families. Another 8 proteins, that is, two proteins in the dehydrogenase family, two phosphofructokinases, two mannosidases, one endochitinase and one uncharacterized protein, were downregulated subsequent to H. parallela larvae treatment, with a highest ratio of 0.611 being observed for mannan endo-14-beta-mannosidase 2.

Carbohydrate concentrations

H. parallela larval attack led to a strongly reduced glucose concentrations in the roots compared to controls (Fig. 4). Although root fructose levels followed the same trend, these changes were not significant. The root sucrose contents in the attacked roots remained unaltered (Fig. 4).