Proteomic analysis of lettuce seed germination and thermoinhibition by sampling of individual seeds at germination and removal of storage proteins by polyethylene glycol fractionation

Abstract

Germination and thermoinhibition in lettuce (Lactuca sativa 'Jianyexianfeng No. 1') seeds were investigated by a proteomic comparison among dry seeds, germinated seeds at 15°C, at 15°C after imbibition at 25°C for 48 h, or at 25°C in KNO3 (all sampled individually at germination), and ungerminated seeds at 25°C, a thermoinhibitory temperature. Before two-dimensional gel electrophoresis analysis, storage proteins (greater than 50% of total extractable protein) were removed by polyethylene glycol precipitation, which significantly improved the detection of less abundant proteins on two-dimensional gels. A total of 108 protein spots were identified to change more than 2-fold (P<0.05) in abundance in at least one germination treatment. Nineteen proteins increasing and one protein decreasing in abundance during germination had higher abundance in germinated 15°C, 15°C after imbibition at 25°C for 48 h, and 25°C in KNO3 seeds than in ungerminated 25°C seeds. Gene expression of 12 of those proteins correlated well with the protein accumulation. Methionine metabolism, ethylene production, lipid mobilization, cell elongation, and detoxification of aldehydes were revealed to be potentially related to lettuce seed germination and thermoinhibition. Accumulation of three proteins and expression of five genes participating in the mevalonate (MVA) pathway of isoprenoid biosynthesis correlated positively with seed germinability. Inhibition of this pathway by lovastatin delayed seed germination and increased the sensitivity of germination to abscisic acid. MVA pathway-derived products, cytokinins, partially reversed the lovastatin inhibition of germination and released seed thermoinhibition at 25°C. We conclude that the MVA pathway for isoprenoid biosynthesis is involved in lettuce seed germination and thermoinhibition.

Figure 1.

Time courses of lettuce seed germination under different conditions. The seeds were imbibed in water at 15°C (black circles) and 25°C (white circles) or in 10 mm KNO₃ at 25°C (diamonds) in 14 h of light (66.3 μmol m⁻² s⁻¹) and then continuous darkness. The ungerminated seeds at 25°C for 48 h were transferred to 15°C for a further germination in 14 h of light (66.3 μmol m⁻² s⁻¹) and then continuous darkness (triangles). Data are means ± se (n = 3).

Figure 2.

PEG precipitation of highly abundant proteins in lettuce seeds. A, Flow chart of PEG fractionation. B, One-dimensional (1-D) SDS-PAGE of unfractionated and fractionated samples. C to E, 2-DE maps of unfractionated and fractionated samples. S4, S8, and S12 are the supernatants of the fractionated samples with 4%, 8%, and 12% PEG, respectively; and P4, P8, and P12 are the pellets of the fractionated samples with 4%, 8%, and 12% PEG, respectively. The boxed areas (a and b) show the regions where storage proteins are dominant and where a significant change was visible after PEG precipitation. Seeds germinated at 15°C were used as material for developing the PEG fractionation method. MW, Molecular mass.

Figure 3.

Differentially accumulated proteins during lettuce seed germination. A, Representative images of the Coomassie Brilliant Blue R-250-stained gel from 25nG seeds. A total of 600 μg of proteins was extracted, separated by 2-DE, and visualized with Coomassie Brilliant Blue R-250. The dry seeds, germinated 15G, 25/15G, and 25nG seeds, and ungerminated 25G seeds were subjected to 2-DE and compared with each other. Each spot (indicated by a red arrow and a number) showing a significant change of more than 2-fold (P < 0.05) in at least one treatment was considered to be differentially accumulated. MW, Molecular mass. B, Functional distribution of differentially accumulated proteins during imbibition in different conditions. Proteomes of the germinated 15G and 25nG seeds and ungerminated 25G seeds were compared with dry seeds, and those of the germinated 25/15G seeds were compared with ungerminated 25G seeds. The number of protein spots whose abundance increased and decreased during imbibition under different conditions is given above each column. C, Venn diagram comparison of proteome changes of seeds during imbibition in different conditions. Protein spots increasing in abundance were most similar among germinated 15G, 25nG, and 25/15G seeds (boxed number), while those decreased in abundance were most similar among germinated 15G and 25nG and ungerminated 25G seeds (boxed number).

Figure 4.

Expression of genes encoding the potentially important proteins for seed germination and thermoinhibition. mRNA samples were extracted from dry seeds (0 h), seeds imbibed in water at 15°C (15G) and at 25°C (25G), at 15°C after imbibition at 25°C for 48 h (25/15G) or in 10 mm KNO₃ at 25°C (25nG) for 6, 12, and 24 h, in ungerminated 25G seeds at 48 h of imbibition (shown as >24 on the horizontal axes), and from germinated 15G, 25/15G, and 25nG seeds (shown as >24 on the horizontal axes). Selected candidate proteins (Table I) and their encoding genes are as follows: A to C, SAM (spot 682, SAM1a; spot 707, SAM1b; spot 713, SAM2); D, LOX2 (spot 126); E, predicted AACT1 (spot 783); F, BIPM (spot 298); G, ICL (spot 290); H, CNP21 (spot 1,243); I, ACOx (spot 1,070); J, predicted GPS2 (spot 807); K, ADF2 (spot 1,431); L, ADH1 (spot 424); M, predicted probable ALDO-KETO REDUCTASE 2-LIKE (AKR2; spot 898); N, ANN1 (spot 981); O, ATF (spot 420); and P, CVS (spot 800). Note that different scales are used along the y axis in the different parts. Data are means ± se (n = 3).

Figure 5.

Accumulation of proteins and expression of genes related to the MVA pathway for isoprenoid biosynthesis. Protein accumulation and gene expression are shown to the left and right of the MVA pathway, respectively. Dry, ungerminated 25G, and germinated 15G, 25/15G, and 25nG seeds were subjected to 2-DE analysis. Three proteins in the MVA pathway, predicted AACT1 (A; spot 783), HMGS2 (C; spot 650), and MDPC2 (H; spot 632), were identified to be differentially accumulated by the 2-DE analysis. Accumulation of those proteins in each treatment is arrayed from left to right as dry (D), 25G, 15G, 25/15G, and 25nG. The protein accumulation level was calculated as in Table I. Genes encoding MVA pathway enzymes, including three AACTs (B), two HMGSs (D), three HMGRs (E), one MK (F), two PMKs (G), and two MDPCs (I), in lettuce were selected for gene expression analysis. mRNAs were extracted from 15G, 25G, 25/15G, or 25nG seeds at 24 HI (white bars), from ungerminated 25G seeds at 48 HI, and from germinated 15G, 25/15G, and 25nG seeds (black bars labeled >24 HI at top). Lovastatin is an inhibitor of HMGR in the MVA pathway. The gene expression data are arrayed from left to right as 25G, 15G, 25/15G, and 25nG seeds. Data are plotted as means ± se (n = 3). Note that the gene expression of HMGR3, PMK1, PMK2, and MDPC1 was down-regulated during imbibition under all conditions (<1 on the y axis).

Figure 6.

Effect of lovastatin (an inhibitor of HMGR), lovastatin plus ABA or cytokinins, and cytokinins on seed germination. A, Germination of seeds at 15°C in response to increasing concentrations of lovastatin. B, Germination of seeds at 15°C in response to the application of 10 μm ABA (A10) and 10 μm ABA plus 10 μm lovastatin (A10+L10) or 100 μm lovastatin (A10+L100). C, Germination of seeds at 15°C in response to the application of 100 μm lovastatin plus 50 μm kinetin (KT), 6-benzylaminopurine (6-BA), or zeatin (ZT). Fifty micromolar cytokinins is an optimum concentration for seed germination (other concentrations are not shown). D, Germination of seeds at 25°C in response to the application of 50 μm KT, 6-BA, and ZT. The dotted lines in B and C show the time course of germination without the application of chemicals at 15°C. The insets in A to C show the germination rate (inverse of time to reach 50% germination [1/t50]) in different germination conditions. Data are plotted as means ± se (n = 3). Where the se is not visible, it was smaller than the symbol.