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 KNO 3 (all sampled individually at germination), and ungerminated seeds at 25°C, a thermoinhibitory temperature. Before twodimensional 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 KNO 3 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.Seed germination is a complex process that is influenced by many environmental factors, such as light, temperature, and moisture (Bewley et al., 2013). Temperature is the most important environmental factor in regulating seed germination. For a given species, there is an optimal temperature or temperature range for seed germination, below and above which seed germination will be delayed or inhibited. The failure of a seed to germinate at supraoptimal temperatures is commonly called thermoinhibition (Reynolds and Thompson, 1971;Abeles, 1986). In nature, thermoinhibition of seed germination is an adaptive regeneration strategy for the seeds of winter annual plants, which prevents the seeds germinating during the summer when the conditions are not appropriate for seedling growth (Baskin and Baskin, 1998;Allen et al., 2007). However, thermoinhibition often creates a problem for crop production. It causes delayed and poor germination of crop species with low optimal temperatures for seed germination and thus limits the time and region for crop cultivation. In the case of lettuce (Lactuca sativa) seeds, which exhibit thermoinhibition at 25°C to 30°C depending on variety and production environment (Guzman et al., 1992...

Mitochondrial Proteome Studies in Seeds during Germination

“…Several different approaches have been applied to monitor the on-going changes in the abundance of mitochondrial proteins during seed germination: (i) a direct study of the global mitochondrial proteome variations using isolated organelles from germinating seeds [12,23]; (ii) a targeted approach to study specific mitochondrial proteins in isolated organelles from germinating seeds [11,26]; (iii) an indirect approach to describe the changes of mitochondrial proteomes in germinating seeds, using total seed protein extracts to identify and measure mitochondrial proteins [13]; (iv) an indirect study with the aim to describe the variations in total proteome in germinating seeds, in which mitochondrial proteins have been detected among many other proteins [16,27,28,29,30] (Figure 1). …”

“…The obtained quantitative protein maps deliver a lot of information regarding the intactness of the protein sample, differences in abundance between two or more biological conditions, protein variants differing in molecular weight and/or pI as well as characterization of post-translational modifications, such as carbonylation, phosphorylation, glycosylation, acetylation, and methylation [31]. 2D-gel electrophoresis has been used to study changes in abundance in the total seed proteome during germination of Arabidopsis, pea, rice, and lettuce seeds [27,28,29,30] (Table S1). In the studies performed by Howell et al [12,23] two-dimensional separation of mitochondrial proteins isolated from rice embryos was applied to examine the effects of oxygen on mitochondrial biogenesis during rice germination (Table S1).…”

“…Miernyk and Hajduk [18] provided a short overview of various possible strategies to reduce storage proteins in a given seed protein sample. Recently, a removal of storage proteins by polyethylene glycol fractionation was utilized successfully in lettuce seeds, improving the detection of less-abundant proteins on 2D-PAGE gels [30]. …”

“…The dynamics of mitochondrial protein abundance in germinating seeds has been observed in many plant species, such as Oryza sativa (rice) [12,23,26,29], Pisum sativum (pea) [28], Arabidopsis thaliana [13,16,27,49], Lactuca sativa (lettuce) [30], and Zea mays (maize) [11]. The list in Table S1 highlights seed mitochondrial proteins, which have shown changes in abundance at different stages during the course of germination, and have been either identified in global proteomic analyses (2DE, MS, iTRAQ) or examined using the targeted proteomic approach (WB, SRM) in different plants.…”

“…While catalase 3 has been shown to increase in abundance in two different experimental set-ups, we found an opposite behavior of superoxide dismutase, being up and down regulated. However, the observed discrepancies among the MSD abundances appear to be due to the differences in the studied plant species and/or germination conditions [12,16,28,29,30]. It has been suggested that successful germination arises from the activity of ROS molecules that function in a certain oxidative window as signaling molecules without harmful consequences for the cell [53].…”