Physiological and protein profiling analysis provides insight into the underlying molecular mechanism of potato tuber development regulated by jasmonic acid in vitro

Abstract

Background: Jasmonates (JAs) are one of important phytohormones regulating potato tuber development. It is a complex process and the underlying molecular mechanism regulating tuber development by JAs is still limited. This study attempted to illuminate it through the potential proteomic dynamics information about tuber development in vitro regulated by exogenous JA.

Results: A combined analysis of physiological and iTRAQ (isobaric tags for relative and absolute quantification)-based proteomic approach was performed in tuber development in vitro under exogenous JA treatments (0, 0.5, 5 and 50 μΜ). Physiological results indicated that low JA concentration (especially 5 μM) promoted tuber development, whereas higher JA concentration (50 μM) showed inhibition effect. A total of 257 differentially expressed proteins (DEPs) were identified by iTRAQ, which provided a comprehensive overview on the functional protein profile changes of tuber development regulated by JA. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis indicated that low JA concentration (especially 5 μM) exhibited the promotion effects on tuber development in various cellular processes. Some cell wall polysaccharide synthesis and cytoskeleton formation-related proteins were up-regulated by JA to promote tuber cell expansion. Some primary carbon metabolism-related enzymes were up-regulated by JA to provide sufficient metabolism intermediates and energy for tuber development. And, a large number of protein biosynthesis, degradation and assembly-related were up-regulated by JA to promote tuber protein biosynthesis and maintain strict protein quality control during tuber development.

Conclusions: This study is the first to integrate physiological and proteomic data to provide useful information about the JA-signaling response mechanism of potato tuber development in vitro. The results revealed that the levels of a number of proteins involved in various cellular processes were regulated by JA during tuber development. The proposed hypothetical model would explain the interaction of these DEPs that associated with tuber development in vitro regulated by JA.

Keywords: Differentially expressed proteins; Jasmonic acid; Potato; Proteome; Tuber development; iTRAQ.

Fig. 1

Effects of exogenous JA on tuber tuberization in vitro. A Tuber morphology, B Tuberization (number of tubers per stolon), C Tuber diameter, D Tuber fresh weight, E Tuber dry weigh. Data are presented as means ± SE for three independent experiments. Different letters indicate that the means differ significantly (p < 0.05) according to Duncan’s multiple range test

Fig. 2

Effect of exogenous JA on the starch (A), reducing sugars (B) and sucrose (C) content of tubers in vitro. Data are presented as means ± SE for three independent experiments. Different letters indicate that the means differ significantly (p < 0.05) according to Duncan’s multiple range test

Fig. 3

Effect of exogenous JA on the H₂O₂ content and antioxidant enzyme activity of tubers in vitro. After 40 days culture, the H₂O₂ content (A), SOD activity (B), APX activity (C) and CAT activity (D) of tubers were measured. Data are presented as means ± SE for three independent experiments. Different letters indicate that the means differ significantly (p < 0.05) according to Duncan’s multiple range test

Fig. 4

Functional classification, cluster analysis and distribution density of JA-responsive DEPs during tuber development in vitro. A The pie chart showed nine protein function categories of DEPs. B A heat-map displayed the differential expression patterns of DEPs in different functional categories. All the protein names and information of heatmap were listed in the Additional file 4: Table S4. C A violin plot displayed the protein expression distribution density of DEPs in different functional categories. The gray dotted line was the average value of protein expression. Pm, metabolism; Pb, protein biosynthesis and degradation; Pt, transcription and translation; Pd, cellular defense; Pg, signaling; Pr, transport, Pa, cell cycle and structure; Po, miscellaneous; Pu, unknown

Fig. 5

The expression pattern of JA-responsive DEPs in different function categories. A The optimal number of clusters was chosen by maximizing the Calinski-Harabasz index (Calinski) of DEPs. The black dashed line indicates the best number of K-median clusters. B The principal component analysis of DEPs distribution in two clusters. The red area represents cluster 1, and the blue area represents cluster 2. C All the DEPs were grouped into two clusters based on the similar expression pattern according to K-median clustering. D The function categories of DEPs in each cluster with similar expression pattern. Pm, metabolism; Pb, protein biosynthesis and degradation; Pt, transcription and translation; Pd, cellular defense; Pg, signaling; Pr, transport, Pa, cell cycle and structure; Po, miscellaneous; Pu, unknown

Fig. 6

The GO term and KEGG pathway enrichment of JA-responsive DEPs during tuber development in vitro. A The GO categorization of DEPs. Three GO sources (BP, biological process; CC, cellular component; MF, molecular function) were represented by red, blue, and green spots, respectively. B The KEGG pathway annotation of DEPs. The number in the spot represents protein number

Fig. 7

Interaction network of JA-responsive DEPs during tuber development in vitro. A The key node proteins in the interaction network were analyzed by Maximal Clique Centrality (MCC) algorithm. The ascending order STRING score is colored from light green to dark blue. The top ten proteins with MCC scores are sorted from lowest to highest and colored from yellow to red. B The interaction of DEPs with different functions. The corresponding functional categories of interacting proteins are represented by the ribbons with different colors in the chord diagram. C The expression correlation of interacting proteins with different functions. The red line indicates a positive relation (p < 0.05), and the blue line indicates a negative relation (p < 0.05). Ts, transcription; Tl, translation; Mc, carbohydrate metabolism; Ma, amino acid metabolism; Me, energy metabolism; Ml, phospholipid; Mp, inorganic phosphate ion metabolism; Mj, JA metabolism; Mu, nucleotide metabolism; Mo, other metabolism; Pe, proteases and peptidases; Pc, protein folding; Pr, transport and channel; Pg, signaling; Pa, cellular structure; Pd, defense; Pi, protease inhibitors; Ps, storage; Po, other proteins

Fig. 8

Immunoblot analysis of key proteins in JA biosynthesis and regulatory pathway during tuber development. The immunoblot expression and iTRAQ expression of COI1 (A), HSP90 (B), and LOX2 (C) were analyzed under different JA concentration. Data are presented as means ± SE for three independent experiments. In order to save antibody and chromogenic reagent, the membranes were cut into strips using the molecular weight standard as a guide after transfer to a PVDF membrane. The first membrane was cut just 1 cm above and below the 70 KDa molecular weight marker and used for COI1 immunoblot. The second membrane was cut just between the 70 KDa and 100 KDa molecular weight markers and used for HSP90 immunoblot. The third membrane was cut just 1 cm above and below the 100 KDa molecular weight marker and used for LOX2 immunoblot. The original gels and blot strips are presented in Additional files 5, 6, 7 and 8: Fig. S1-4

Fig. 9

The JA-responsive DEPs involved in cell wall and cytoskeleton composition during tuber development in vitro. The heat-map presented the expression change of these DEPs. The significance of t-test was presented by “*” (p < 0.05). The green band indicated the corrected p-value (P_adj < 0.05, one-way ANOVA analysis of variance followed by Bonferroni correction for multiple comparison) was mapped as an annotation of heatmap. ADF, actin depolymerizing factor; MAP65-1a, microtubule-associated protein 65-1a; SnRK2.4, SNF1-releted protein kinases 2.4; SPS, sucrose-phosphate synthase; TCTP, translationally-controlled tumor protein; UAM, alpha-1,4-glucan-protein synthase; UGE4, UDP-glucose 4-epimerase; V-SNARE, soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors

Fig. 10

The JA-responsive DEPs involved in primary carbon metabolism during tuber development in vitro. The heat-map presented the expression change of these DEPs. The significance of t-test was presented by “*” (p < 0.05). The green band indicated the corrected p-value (P_adj < 0.05, one-way ANOVA analysis of variance followed by Bonferroni correction for multiple comparison) was mapped as an annotation of heatmap. ACLY, ATP citrate synthase; ENO, phosphopyruvate hydratase; FBA, fructose-bisphosphate aldolase; G6PDH, glucose-6-phosphate dehydrogenase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IDH, isocitrate dehydrogenase; MDH, malate dehydrogenase; PDH, pyruvate dehydrogenase; PEPC, Phosphoenolpyruvate carboxylase; PEPCK, phosphoenolpyruvate carboxykinase; PFP, pyrophosphate: fructose 6-phosphate phosphotransferase; PGAM, phosphoglycerate mutase; PGK1, phosphoglycerate kinase 1; PGM, phosphoglucomutase; PK, pyruvate kinase; Rubisco, ribulose 1,5-bisphosphate carboxylase; TAL, transaldolase; TKT, transketolase

Fig. 11

The JA-responsive DEPs involved in protein biosynthesis, degradation and assembly during tuber development in vitro. The heat-map presented the expression change of these DEPs. The significance of t-test was presented by “*” (p < 0.05). The green band indicated the corrected p-value (P_adj < 0.05, one-way ANOVA analysis of variance followed by Bonferroni correction for multiple comparison) was mapped as an annotation of heatmap. BIP, luminal binding protein; CNX, calnexin; GRP94, glucose-regulated protein 94; EIF2, eukaryotic translation initiation factor 2; EIF6, eukaryotic translation initiation factor 6; HnRNP A1, heterogeneous nuclear ribonucleoprotein A1; HnRNP G, heterogeneous nuclear ribonucleoprotein G; HSP20, heat shock protein 20; HSP90A, heat shock protein 90A; HYOU1, hypoxia up-regulated protein 1; NOP58, MAR-binding protein NOP58; PSMB6, proteasome subunit β type 6; RAN, GTP-binding nuclear protein; RPN1, proteasome subunit RPN1; RPT2, proteasome subunit RPT2; RP-L3e, ribosomal protein L3e; RP-L7Ae, ribosomal protein L7Ae; RP-L10Ae, ribosomal protein L10Ae; RP-L17e, ribosomal protein L17e; RP-L18e, ribosomal protein L18e; RP-S2e, ribosomal protein S2e; RP-S23e, ribosomal protein S23e; RP-S24e, ribosomal protein S24e; SF3b3, splicing factor 3b subunit 3; UAP56, DEAD-box ATP-dependent RNA helicases; SAR1, secretion-associated RAS superfamily 1; DKC1, Dyskerin 1