Plant Molecular Biology Reporter https://doi.org/10.1007/s11105-018-1115-x
ORIGINAL PAPER
Comparative Proteomics Reveals the Mechanisms Underlying Variations in Seed Vigor Based on Maize (Zea mays L.) Ear Positions Yan Li 1
&
Haibin Qu 1 & Pengyu Zhu 1 & Kemei Su 1 & Chunqing Zhang 1
# Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract Seed vigor is influenced by seed position in plant. However, current understanding of its underlying mechanism is limited. In this study, we used isobaric tags for relative and absolute quantitation technique to study the comparative proteomes between middle seeds (with higher vigor) and top seeds of maize (Zea mays L.) ears at 0 h, 24 h, and 48 h of imbibition. A total of 159 differentially accumulated proteins were identified. Among these, the largest number of proteins was from the functional categories of Disease/Defense and Metabolism. Compared with top seeds, most of the differentially accumulated proteins of Protein Synthesis and Energy showed higher accumulation in middle seeds at 0 h and 24 h of imbibition, but lower accumulation at 48 h of imbibition. Seed water absorption activates metabolic processes. The water content of middle seeds was significantly lower than that of top seeds at between 12 h and 30 h of imbibition, but energy production would be higher in the middle seeds at 24 h of imbibition. Meanwhile, tonoplast intrinsic proteins 3.1 and 3.2, which mediate water inflow into protein storage vacuoles, then activating enzymes involved in reserve mobilization, showed higher accumulation in middle seeds at 24 h of imbibition. In addition, our data also showed middle seeds may suffer less fungal damages. Our results contribute to understanding the mechanisms underlying the effects of growth position on seed vigor. Keywords Aquaporins . Fungal resistance . Proteomics . Seed position . Seed vigor . Zea mays
Introduction Seed vigor is an important indicator of seed quality. Highvigor seeds germinate rapidly and produce strong and resistant Yan Li and Haibin Qu contributed equally to this work. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11105-018-1115-x) contains supplementary material, which is available to authorized users. * Yan Li
[email protected] * Chunqing Zhang
[email protected] Haibin Qu
[email protected] Pengyu Zhu
[email protected] Kemei Su
[email protected] 1
State Key Laboratory of Crop Biology, Agronomy College, Shandong Agricultural University, Taian 271018, China
seedlings (Finch-Savage and Bassel 2016), which are essential to agricultural production. Seed vigor gradually improves with seed development (Finch-Savage and Bassel 2016). Developmental environments, including the position of the seeds on the plant, significantly influence seed vigor (Gutterman 2000). The maize (Zea mays L.) ear develops from the female inflorescence by double fertilization. Each ear contains hundreds of ovules, each of which produces a strand of maize silk (an elongated style) from the tip that eventually emerges from the end of the ear and then gets pollinated (Kiesselbach 1999). The silks from the middle and bottom sections of the ear emerge earlier than those from the top section and are pollinated earlier, which render the top seeds the weakest sinks for assimilates (Shen et al. 2018; Turc et al. 2016). Mondo and Cicero (2005) reported that the bottom and middle seeds of the maize ear exhibited higher vigor than the top seeds. To our knowledge, however, no reports involving the associated proteomes are available. Water is the primary requirement for seed germination (Bewley et al. 2013). The entry of water into plants involves three routes: the apoplastic pathway, symplastic pathway, and transcellular water transport (Postaire et al. 2007). Aquaporins are involved in water inflow to plant organs by transcellular
Plant Mol Biol Rep
water transport (Obroucheva and Sin’kevich 2010), which may mediate a fine temporal and spatial control of water transport (Maurel et al. 2008). Generally, water is transported into cells via plasmalemma intrinsic proteins (PIPs), then some of water inflows into vacuoles through tonoplast intrinsic proteins (TIPs) (Tyerman et al. 1999). However, Gattolin et al. (2011) localized TIP3;1 and TIP3;2 to both tonoplast and plasma membrane of Arabidopsis embryo, suggesting that water can also enter cells via TIP3s. Nevertheless, TIP3s are generally considered as marker proteins located at protein storage vacuole (PSV) membranes (Hunter et al. 2007; Jauh et al. 1999), which gradually accumulate during seed development and maturation (Melroy and Herman 1991), are abundant in dry seeds and seeds during early imbibition, and then vanish when vacuoles recovering from PSVs (Novikova et al. 2014). In addition, TIP3.1 is specifically induced in maize seeds by the germination-promoting substance KAR1 (3methyl-2H-furo[2,3-c]pyran-2-one), indicating that TIP3.1 plays important positive roles in maize seed germination (Soós et al. 2010). When seed water content is reduced to 20%, membrane integrity is lost, disrupting the construction of the plasma membrane of dry seeds (Simon 1974). Therefore, when seeds are immersed in water or planted in moist soils, a variety of solutes such as sugars, ions, amino acids, and proteins leak out (Bewley et al. 2013), potentially resulting in bacteria and fungi growth in the medium surrounding the seeds, which may then invade the seeds, ultimately leading to pre-emergence mortality of seeds (Simon 1974). Campo et al. (2004) reported changes in the maize embryo proteome after Fusarium verticillioides infection and found that proteins involved in protein synthesis, folding, and stabilization and ones involved in oxidative stress tolerance such as chitinases and glucan endo-1,3-beta-glucosidases are induced. Cordero et al. (1994) also found that infection of maize seeds with Fusarium moniliforme induces the expression of chitinases and glucan endo-1,3-beta-glucosidases. Germination is an energy-consuming process. Glycolysis works under both aerobic and anaerobic conditions to produce pyruvate. As the seed coat and other surrounding structures restrict the supply of O2 to embryos during imbibition, pyruvate was then reduced further to lactic acid, or ethanol plus CO 2 , whereas after radicle emergence, pyruvate was completely oxidized to CO2 and water via tricarboxylic acid (TCA) cycle, producing more ATP molecules (Bewley et al. 2013). Nevertheless, the glycolysis pathway may have a greater effect on seed vigor than TCA cycle (Wang et al. 2015). In high-vigor maize seeds, glyceraldehyde-3-phosphate dehydrogenase, cytosolic 3 (Wang et al. 2013), and fructosebisphosphate aldolase (Wu et al. 2011) are highly accumulated, while in aged low-vigor maize seeds, phosphoglucomutase, 3-phosphoglycerate kinases, and triosephosphate isomerase are downregulated significantly (Xin et al. 2011),
while malate dehydrogenase, a key enzyme in TCA cycle, is upregulated by artificial aging, suggesting that TCA cycle is disturbed by the treatment (Xin et al. 2011). In addition, oxidative phosporylation also plays an important role in ATP production in dry and imbibing maize seeds (Logan et al. 2001). ATP synthase subunits were found upregulated in high-vigor maize seeds (Wang et al. 2013) and down regulated in aged seeds (Xin et al. 2011). Proteomics is a powerful tool for studying complex traits. At present, proteomics have numerous applications in seed vigor research. In these studies, most of the seeds exhibit different levels of vigor mainly through artificial accelerated aging (Chu et al. 2012; Xin et al. 2011) or priming (Catusse et al. 2011). In addition, Wang et al. (2013) reported proteomic comparison between maize seeds subject to maturation drying and premature imposed drying and revealed a potential role of maturation drying in preparing proteins for seed vigor. In this study, we used middle and top seeds of naturally dried maize ears as materials, and the difference in seed vigor is based on changes in seed developmental processes due to growth positions. We comparatively studied the physiology and proteome of middle and top seeds at 0 h, 24 h, and 48 h of imbibition. Our results contribute to understanding the mechanisms underlying the effect of growth position on seed vigor.
Materials and Methods Plant Materials We selected three Chinese maize hybrids, ZD958, XD20, and JX1, as experimental materials. The top and middle seeds of ZD958 were used for the proteomics assay, and the three imbibition treatments were for 0 h, 24 h, and 48 h.
Germination Test Fine sand (about 0.35 mm in diameter) was used as a germination bed, and the germination test was conducted under the conditions of the sand bed water content of 38.5%, the temperature of 25 °C ± 1 °C, and the illumination for 24 h a day. The bed thickness was 4 cm, the seed embryo was placed facing upwards, and the overlaying sand thickness was 2 cm. Four replicates, with 100 seeds for each, were used in the test. Germination was considered to have occurred when the coleoptile had broken through the overlaying sand layer, and the germinated seeds were enumerated daily. At the end of the seventh day, 30 normal seedlings of each replicate were randomly selected for the fresh weight (W). The remaining seeds were stripped off, and then the fresh weight (W) was measured. The following formulas were used to calculate vigor index (VI), germination index (GI), and fresh weight per plant (S):
Plant Mol Biol Rep
VI ¼ GI S GI ¼ ∑ðGt=DtÞ S ¼ W=30 where Gt is germination number per day, Dt is germination days, and W is fresh weight of 30 normal seedlings on the seventh day of germination (g).
Hundred-Grain Weight Determination One hundred maize seeds were randomly counted to determine the 100-seed weight, with eight replicates. When the coefficient of variation was less than four, the eight measured values were deemed valid, and the average of the eight replicates was taken as the measured 100-grain weight (W0). The water content of the maize seeds was then measured using a capacitive water analyzer (PM888, Kett, Japan) with a 2% difference between the two replicates being effective, and the average of the two replicates was considered as the seed water content (C0). The following formula was used to calculate the 100-grain weight (W) at 13% water content: W ¼ W 0 ð1−C 0 Þ=ð1−13%Þ where W0 is the measured 100-grain weight (g), and C0 is seed water content (%). Seven days after sowing in a sand bed, 30 seedlings of ZD958 were randomly selected, and eight replicates were taken. The remaining seeds were stripped off and dried at (105 ± 2)°C until constant weight (m0). Seed water content (C0′) was measured as earlier described, and post-germination 100-grain weight (Wa) was calculated using the following formula: 0 0 W a ¼ W 0 1−C 0 =ð1−13%Þ 0
W 0 ¼ m0 =30 100 where m0 is the dry weight of the remaining seeds after being stripped off from 30 normal seedlings after germination (g), W 0′ is the measured 100-grain weight after germination (g), and C 0′ is the measured seed water content after the remaining seeds were dried (%).
Seed Specific Gravity Determination Approximately 40 mL of distilled water was added into a 100-mL graduated cylinder, to which about 30 g of maize seeds of ZD958 were added. When the liquid level in the graduated cylinder had stabilized, the volume was recorded and the process repeated in triplicate. Seeds specific gravity (Sp) was calculated as follows: S p ¼ W 0 =ðV 2 −V 1 Þ
where W0 is the weight of the seeds added to the graduated cylinder (g), V1 is the volume of distilled water (mL), and V2 is the total volume (mL) of distilled water and seeds added to the graduated cylinder.
Protein Extraction, Digestion, iTRAQ Labeling, and Tandem Mass Spectrometry Analysis Mature ZD958 whole seeds from middle and top sections of the ears imbibed for 0 h, 24 h, and 48 h were used as experimental materials for proteomics assay, two biological replications with 30 seeds for each. After imbibition, all the seeds were sent to Beijing Genomics Institute (BGI, Shenzhen, China) for further processing. All the 30 seeds were immersed in liquid nitrogen and ground to a fine powder. After thoroughly mixing, 100 mg of fine powder were weighed, mixed with 1 mL of lysis buffer (5 mM Tris-HCl, pH 7.4, 1 mM PMSF, 2 mM EDTA, 10 mM DTT, and 1% Triton X-100), and then subjected to ultrasonic vibrations for 15 min. The subsequent experiment was performed as described by Li et al. (2015).
Protein Identification and Analysis The MS data were performed using the Proteome Discoverer 1.3 software (Thermo Fisher Scientific, San Jose, CA, USA). Protein identification and relative abundance quantitation were processed using Mascot 2.3.02 (Matrix Science, London, UK). The analysis was conducted with maize inbred line B73 protein database (http://ftp.maizesequence.org/, ZmB73_5b_FGS, 58,129 sequences). The search parameters were as follows: Type of search: MS / MS Ion search; Trypsin digestion; Fragment Mass Tolerance: ± 0.1 Da; Mass Values: Monoisotopic; Variable modifications: Gln- > pyro- Glu (Nterm Q), Oxidation (M), iTRAQ8plex (Y); Peptide Mass Tolerance: ± 0.05 Da; Default instrument type; Max Missed Cleavages: 1; Fixed modifications: Carbamidomethyl (C), iTRAQ8plex (N- term), iTRAQ8plex (K). A protein was regarded as differentially accumulated when the protein abundance differed by > 1.2-fold and the P value was < 0.05 for seeds in the different sections of maize ear. DAPs were classified into different functional classes according to Bevan et al. (1998). DAPs were classified according to Gene Ontology (http://www.geneontology.org).
Conductivity Measurement Approximately 30 g of intact maize seeds of ZD958 were washed with distilled water, and after wiping off the surface water, the seeds were placed into a volumetric flask containing 50 mL of distilled water. Changes in conductivity were measured at 0 h, 6 h, 12 h, 24 h, 48 h, and 72 h with a highprecision desktop conductivity meter (Thermo Scientific,
Plant Mol Biol Rep
Orion Star A215, USA) in triplicate. Seed leachate conductivity (Ec) was calculated as follows: E c ¼ E0 =W 0 ; where Ec is the seed conductivity (μS−1 cm−1 g−1), E0 is the measured conductivity (μS−1 cm−1), and W0 is the seed weight (g).
Seed Water Intake Determination The seed water intake test was carried out in the sand bed, which was the same as the above germination test, three replications with 15 intact seeds of ZD958 for each. The total weight of 15 seeds (wiping off surface water) after 0 and 12 h of imbibition was measured, and then placed in a sand bed to allow imbibition. The total weight of the seeds was then measured every 2 h for a total of 30 h of imbibition. Seeds water content of 0 h of imbibition was determined in accordance with the method described in section “Hundred-Grain Weight Determination.” The remaining water content was calculated according to the following formula: C t ¼ ðW t −W 0 þ W 0 C 0 Þ=Wt 100% where Ct is the water content of seeds at t h of imbibitions, Wt is the total weight of seeds at t h of imbibitions, W0 is the total weight of seeds at 0 h of imbibition, and C0 is the water content of seeds at 0 h of imbibition. Two-hour-on-two-hour water uptake growth rate is calculated according to the following formulas: Gt % ¼ ðI t −I t−2 Þ=I t−2 100% I t ¼ ðW t −W 0 Þ=W 0 100% where Gt is the two-hour-on-two-hour water uptake growth rate, It is the water intake rate of seeds at t h, Wt is the total weight of seeds at t h of imbibition, and W0 is the total weight of seeds at 0 h of imbibition.
Enzyme Activity Determinations Thirty maize seeds were ground under liquid nitrogen condition. Five grams ground seed sample was mixed with 20 mL extract solution (KCl 2.0 M, Tris 0.2 M, EDTA 0.02 M, pH 8.0) for 5 min, and then the mixture was filtered. The whole process took place under the ice bath. The filtrate was centrifuged at 6500 g for 30 min. Then, 10 mL supernatant was diluted to 25 mL. Alcohol dehydrogenase activity was then determined according to the description of Hanson and Jacobsen (1984). Alpha-amylase activity was determined by the DNS method (Bernfeld 1955). Thirty seeds were ground and then were mixed with 20 mL distilled water. All the homogenate was
transferred into a 50-mL centrifuge tube and then diluted to 50 mL. After extracting at room temperature for 20 min, the solution was centrifuged at 3000g for 10 min. The supernatant was diluted to 100 mL. Then, alpha-amylase activity was determined according to the methods of Bernfeld (1955). As for catalase and peroxidase activity, 30 seeds were put into a precooled mortar, then added with 15 mL precooled phosphate buffer (4 °C, PH 7.8). Under ice bath condition, seeds were ground into homogenate. All the homogenate was transferred into a 50-mL centrifuge tube and then diluted to 50 mL. Then, the solution was centrifuged at 12,000 rpm for 10 min. Then, 10 mL supernatant was diluted to 50 mL. Catalase activity was determined according to the methods of Kato and Shimizu (1987), and peroxidase activity were determined according to the description of Lamikanra and Watson (2001). Three independent biological replicates were analyzed for determining the activity of the four enzymes, with 30 intact seeds of ZD958 for each.
Statistical Analysis All data are obtained at least from three independent biological replicates. All statistical analyses were performed using the IBM SPSS Statistics 19.0 software (SPSS, Chicago, IL, USA).
Results Maize Seeds at the Middle and Bottom Sections of the Ear Produce Stronger Seedlings Seeds from top, middle, and bottom sections (Online Resource 1) of the ears of three maize hybrids, Zhengdan 958 (ZD958), Xundan 20 (XD20), and Jixiang 1 (JX1), were germinated. Vigor index (VI) of the middle or bottom seeds of the maize ear was significantly higher than that of the top seeds (P < 0.01, Fig. 1a), whereas germination index (GI) showed no significant differences among seeds from any of the different sections of the maize ear (Fig. 1b). The difference in VI mainly emanated from the difference in seedling weights (Fig. 1c). In addition, the length of seedlings did not significantly differ (Fig. 1d, e), which indicated that the seedlings produced by the middle and bottom seeds were stronger. To determine the cause for the seedlings of the middle and bottom seeds being more robust, we measured the 100-grain weight of the seeds at different sections of the maize ear. The results showed that the seeds at the bottom part of the maize ear were the heaviest, followed by those at the middle part, and those at the top section were the lightest. The differences between these sections were highly significant (P < 0.01, Fig. 1f), suggesting that seed weight does not explain the observed
Plant Mol Biol Rep Fig. 1 Germination capacity and physical traits of seeds at different maize ear positions. a Vigor index. b Germination index. c Fresh weight per plant. d Root length. e Shoot length. f Hundredgrain weight. g Seed specific gravity. h Hundred-grain weight of ZD958 before and after 7 days after sowing in sand bed. Data represent the means ± SD of three (g), four (a–e) or eight (f, h) replicates, with 100 (a, b, f), 30 (c–e, h) or 30 g (g) seeds each. An ANOVA test followed by Tukey’s post hoc test was performed. Capital letters are used for indicating extremely significant difference (P < 0.01) and lowercase letter for significant difference (P < 0.05)
differences in seedling robustness. Seed specific gravity is closely related to seed vigor (Sung and Delouche 1962). We measured the specific gravity of the seeds at different parts of the ear. The results showed that the specific gravity of the seeds in the middle section of ZD958 and JX1 was significantly higher than that in the top part (P < 0.05), while XD20 exhibited a different pattern (Fig. 1g). The results showed that
specific gravity was positively correlated with stronger seedlings, but was not the primary factor. In addition, the 100-seed weight of the remaining seeds after 7 days of germination was measured. The results demonstrated that about two thirds of the nutrients remained in the top seeds (Fig. 1h), indicating that the weak seedlings from the seeds at top section were not the result of nutrient insufficiency.
Plant Mol Biol Rep
Identification of Differentially Accumulated Proteins (DAPs) To elucidate the mechanisms accounting for the differences in robustness of the seedlings produced by the seeds at different sections of maize ear, the total protein of mature whole seeds was extracted from the middle and top seeds at 0 h, 24 h, and 48 h of imbibition (Online Resource 1). The isobaric tag for relative and absolute quantitation (iTRAQ) technique was used to examine the differences in proteomes between middle and top seeds among the three treatments. At 24 h of imbibition, the seeds were in phase II of water uptake (Logan et al. 2001). At 48 h of imbibition, the radicles had broken through the seed coat, and the radicle length was shorter than the seed length (Online Resource 1). A protein was regarded as a DAP when the protein abundance differed by > 1.2-fold and the P value was < 0.05 in both biological replicates. There were 43, 58, and 85 DAPs between the middle and top seeds of the maize ear at 0 h, 24 h, and 48 h of imbibition, respectively (Fig. 2a). DAPs were divided into different functional classes according to Bevan et al. (1998) (Figs. 2b–d, Online Resources 2–4). Among the three treatments, the largest number of DAPs was from Disease/Defense and Metabolism classes, and also many DAPs were from Energy, Protein Destination and Storage, and Protein Synthesis classes. In addition, in the protein group of higher accumulation in middle seeds, the proportions of DAPs from Energy class at 0 h of imbibition (23.5%) and Metabolism class at 24 h (25.0%) and 48 h (33.3%) of imbibition were respectively the highest. While in the lower accumulation protein group in middle seeds, Disease/Defense class DAPs accounted for the highest proportion at all the three time points, 46.2% in M0/T0, 42.3% in M1/T1, and 22.4% in M2/ T2 respectively. During seed imbibition, the ability of protein synthesis is considered a characteristic feature of seed vigor (Catusse et al. 2011). Four proteins of the Protein Synthesis class were differentially accumulated between middle and top seeds at 0 h and 24 h of imbibition, of which three were highly accumulated in the middle seeds (Fig. 2b, c). In contrast, at 48 h of imbibition, all eight proteins of the Protein Synthesis class less accumulated in the middle seeds (Fig. 2d). Except for one predicted translation initiation factor 2B subunit (GRMZM2G139533_P01) and one elongation factor (GRMZM2G122871_P01), all the DAPs in the class are ribosomal proteins (Online Resources 2–4). Plant ribosomes contain about 70–80 different ribosomal proteins depending on the species (Merchante et al. 2017). Heterogeneous ribosomes selectively translate certain genes (Imai et al. 2008; Shi et al. 2017). The DAPs of the Energy class also showed the same accumulation pattern: most DAPs were highly accumulated in the middle seeds at 0 h (4/6) and 24 h (5/6) of imbibition, while most DAPs had lower accumulation in the middle seeds at 48 h (7/8) of imbibition (Figs. 2b–d, Online Resources 2–4).
Among them, the number of proteins belonging to glycolytic pathways was the largest, accounting for 50.0%, 50.0%, and 37.5% of all the DAPs of Energy class at 0 h, 24 h, and 48 h of imbibition, respectively. In addition, two aquaporins, GRMZM2G037327_P01 (aquaporin TIP3.1) and GRMZM2G103983_P01 (aquaporin TIP3.2), were differentially accumulated. One of them, aquaporin TIP3.2, 5.94 times higher in the middle seeds than in the top seeds at 24 h of imbibition, showed the maximum difference in this study.
Leachate Conductivity of the Top Seeds Was Higher During the drying of seeds, membrane integrity is gradually lost (Simon 1974). When the seeds are imbibed, membranes of viable seeds are gradually repaired, and high-vigor seeds have higher membrane repair efficiency (Hampton and Tekrony 1995). In the present study, we determined the leachate conductivity of the seeds from different maize ear positions. Our results showed that leachate conductivity of the top seeds was significantly higher than that of the middle seeds, and the difference gradually increased with increased imbibition time (Fig. 3), indicating that more solutes were leaked out from the top seeds and that the top seeds showed lower repair efficiency of plasma membrane than the middle and bottom seeds.
Comparison of Water Absorption of Seeds from Different Maize Ear Positions Water absorption is the premise of seed germination (Bewley et al. 2013). To determine the water intake of the middle and top seeds of the ear, seed water contents were measured at different time of imbibition. The results showed that water content of the top seeds was significantly higher than that of the middle seeds at all the time points of imbibition (Fig. 4a). However, after 16 h of imbibition, water uptaking growth rates (two-hour-on-two-hour) of the middle seeds were higher than those of the top seeds, reaching significant level (P < 0.05) at 20 h, 24 h, and 30 h of imbibition (Fig. 4b). The water uptake of phase II stage was related to the decrease of seed water potential caused by limited storage reserves mobilization within the cells that will expand in the embryonic axis (Bewley et al. 2013). The results suggested that the reserves degradation level within the embryonic axis cells during phase II was higher in middle seeds than that in the top seeds.
Enzyme Activity Determinations To validate the proteomic data, enzyme activities of alcohol dehydrogenase (Energy class), alpha-amylase (Metabolism class), catalase, and peroxidase (Disease/Defense class) in seeds at 0 h, 24 h, and 48 h of imbibition were determined. In the proteomic data, alcohol dehydrogenase 1 (GRMZM2G442658_P02) and
Plant Mol Biol Rep
a Number of DAPs
60
Higher accumulation Lower accumulation
50
49 36
40
32 26
26
M0/T0
M1/T1
30 17
20 10 0
b
M2/T2
M0/T0 higher accumulation M0/T0 lower accumulation
Cell structure Disease/defence Energy Metabolism Protein destination and… Protein synthesis Signal transduction Transcription Intracellular transporters Transporters Unclear classification
1
3
12 4
2 2 1
5
1 3
1 1 1 1 2
1 1 1 0
5
10 Number of DAPs
15
M1/T1 higher accumulation
c
M1/T1 lower accumulation 1
Cell structure
6
Disease/defence Energy
11
5
1
Metabolism
8
4
Protein destination and…
3
1
Protein synthesis
3
1 1
Signal transduction Transporters
4
3
1
Unclear classification
5
0
2
4
6 8 Number of DAPs
10
12
M2/T2 higher accumulation M2/T2 lower accumulation
d Cell structure
2 5
Disease/defence
11
1
Energy
7
Metabolism
12
6 4
Protein destination and storage
5
Protein synthesis
8 3
Secondary metabolism Signal transduction
1
Transporters
1
1 4
Unclear classification
5 0
2
Fig. 2 Differentially accumulated proteins (DAPs) identified in this study. a Number of DAPs. b Functional classes of DAPs between middle and top seeds imbibed for 0 h. c Functional classes of DAPs between middle and top seeds imbibed for 24 h. d Functional classes of
4
6 8 Number of DAPs
9 10
12
14
DAPs between middle and top seeds imbibed for 48 h. M, middle seeds; T, top seeds. 0, 1, and 2 mean that seeds were imbibed for 0 h, 24 h, and 48 h respectively
Plant Mol Biol Rep 40.00 Electrical conductivity of seed leachates (µS cm-1 g-1)
Top
Middle
**
35.00 30.00
*
25.00 20.00 * 15.00 10.00 **
*
*
5.00 0.00 0
8
16
24
32
40
48
56
64
72
Soaking period (h)
Fig. 3 Leachate conductivity of middle and top seeds soaked for different hours. Data represent the means ± SD of three replicates, with about 30 g seeds for each. An ANOVA test followed by LSD post hoc test was performed. Double asterisks (**) are used for indicating extremely significant difference (P < 0.01) and single asterisk (*) for significant difference (P < 0.05)
alcohol dehydrogenase 2 (GRMZM2G098346_P01) were highly accumulated in middle seeds at 48 h and 24 h of imbibition, respectively (Online Resources 3–4). Enzyme activities determination showed that the activity of alcohol dehydrogenase of middle seeds was also significantly higher (P < 0.05) than that of top seeds at the two time points (Fig. 5a). Similarly, alphaamylase isozyme 3C (GRMZM2G138468_P01) was highly accumulated in middle seeds both at 24 h and 48 h of imbibition (Online Resources 3–4). And alpha-amylase activity was also higher in middle seeds and reached significant level (P < 0.05) at 48 h of imbibition (Fig. 5b). Catalase isozyme B (GRMZM2G088212_P01) was lower accumulated in middle seeds at 24 h of imbibition in the proteomic data (Online Resources 3), and the activity of catalase was also significantly lower (P < 0.05) in middle seeds at 24 h of imbibition (Fig. 5c). Peroxidase 45 (GRMZM2G080183_P01) was lower accumulated in middle seeds at 0 h of imbibition (Online Resources 2). The activities of peroxidase were also lower in middle seeds, but did not reach significant level at 0 h of imbibition (Fig. 5d).
Discussion Seed vigor is an important trait of evaluating seed quality, which is different at different positions of the plant (Gutterman 2000). So, seed grading is conducted by seed companies to grade seeds with similar vigor for uniform seedlings. But little is known about the mechanisms underpinning the seed vigor differences between seeds located at different positions of the plant. In this study, using middle (high-vigor) and top (low-vigor) seeds of maize ears as materials, we explored the mechanisms of the effects of different growth positions on seed vigor by comparative proteomics. In our view, maize is the ideal plant
to study the effect of the growth position on seed vigor. First of all, seeds located on the same maize ear face almost the same external environmental conditions (light, airing, etc.), and the differences of seed vigor is only affected by internal factors. In addition, unlike plants such as wheat and rice, common maize usually has only one stem and no branches on the ear. So, the factors that influence vigor of seeds located at different maize ear positions are relatively simple. Below we discuss the interesting findings of our work.
Real-Time Seed Vigor of Middle Seeds Should Be Higher Than That of Top Seeds at 24 h of Imbibition, But Lower at 48 h of Imbibition The main metabolic processes, including protein synthesis, occur during phase II, although the net water uptake of seeds is relatively limited (Bewley et al. 2013). The protein synthesis capacity during imbibition of the seed is considered to be a characteristic feature of seed vigor (Catusse et al. 2011). In this study, at 24 h of imbibition, three out of the four DAPs in the Protein Synthesis class exhibited higher accumulation in the middle seeds, while at 48 h of imbibition, all the eight DAPs in the Protein Synthesis class exhibited lower accumulation in the middle seeds (Fig. 2c, d, Online Resources 3–4). Among all the DAPs, only two proteins, elongation factor 1-gamma 2 (GRMZM2G122871_P01) and 60S ribosomal protein L4-2 (GRMZM2G100403_P02), were differentially accumulated both at 24 h and 48 h of imbibition. While, the differentially accumulated patterns of them between middle and top seeds at 24 h and 48 h of imbibition were completely opposite, higher accumulation in middle seeds at 24 h, but lower accumulation at 48 h of imbibition. The results indicated that the ability of the middle seeds to synthesize proteins was higher than that of the top seeds at 24 h of imbibition, whereas the opposite was observed at 48 h of imbibition. The seed germination process consumes a large amount of energy, which was produced by degradation of reserves. The intensity of the energy produced influences seed germination rate and seedling robustness (Bewley et al. 2013). In this study, there were six DAPs of the Energy class between the middle seeds and top seeds at 24 h of imbibition, of which five showed higher accumulation in the middle seeds. While at 48 h of imbibition, seven out of all the eight DAPs of the Energy class were less accumulated in the middle seeds. At 24 h of imbibition, three of the five proteins that were highly accumulated in the middle seeds belonged to glycolytic pathways (GRMZM2G446253_P01, enolase 2; GRMZM2G130440_P02, aldehyde dehydrogenase family 7 member B4; and GRMZM2G098346_P01, alcohol dehydrogenase 2), while the other two, v-type proton ATPase (GRMZM2G101020_P01) and cytochrome c (GRMZM2G032367_P01), belonged to oxidative phosphorylation pathway. Only one enzyme belonging to the TCA cycle was
Plant Mol Biol Rep
a 45.00 Top
Middle
40.00 35.00 Seed water content (%)
Fig. 4 Water absorption of middle and top seeds. a Seed water content of middle and top seeds imbibed for different hours. b Two-hour-on-two-hour water uptaking growth rate of middle and top seeds imbibed for different hours. Data represent the means ± SD of three replicates, with 15 seeds each. An ANOVA test followed by LSD post hoc test was performed. Double asterisks (**) are used for indicating extremely significant difference (P < 0.01) and single asterisk (*) for significant difference (P < 0.05)
**
**
**
**
**
**
14
16
18
20
22
**
*
*
24
26
28
*
30.00 25.00 20.00 15.00 10.00
*
5.00 0.00 0
2
4
6
8
10
12
30
Imbibition time (h)
b 10.00
Top
Middle
Water uptaking growth rate (two-hour-on-two-hour, %)
9.00 8.00 7.00
*
6.00 5.00
*
4.00
*
3.00 2.00 1.00 0.00 14
16
differentially accumulated (GRMZM2G039251_P01, succinylCoA ligase [ADP-forming] subunit alpha-1, mitochondrial), and the accumulation level in the middle seeds was lower than that in the top seeds. At 48 h of imbibition, except for alcohol dehydrogenase 1 (GRMZM2G442658_P02), which was highly accumulated in the middle seeds compared to that in the top seeds, the other seven proteins of Energy class accumulated less in the middle seeds than in the top seeds. Among them, three enzymes belong to the glycolysis pathway (GRMZM2G446253_P01, enolase 2; GRMZM2G178958_P01, leucine aminopeptidase 1; and GRMZM2G161868_P01, ketol-acid reductoisomerase, chloroplastic), and three enzymes belong to the TCA cycle (GRMZM5G858454_P02, aconitate hydratase, cytoplasmic; GRMZM2G033226_P01, ATP-citrate synthase beta chain protein 2; and GRMZM2G015132_P01, dihydrolipoyllysine-
18
20 22 24 Imbibition time (h)
26
28
30
residue acetyltransferase component 3 of pyruvate dehydrogenase complex), and one enzyme is involved in ATP hydrolysis (GRMZM2G093347_P01, adenine phosphoribosyltransferase 1). In addition, consistent with the oxidative phosphorylation pathway proteins mentioned above, two carrier proteins belonging to this pathway, GRMZM2G105571_P01 (ADP, ATP carrier protein) and GRMZM2G015401_P01 (phosphate carrier protein), also showed higher accumulation at 24 h of imbibition and lower accumulation at 48 h of imbibition in middle seeds. Altogether, these results indicated that the middle seeds produced more energy at 24 h of imbibition, while the top seeds showed a higher level of energy metabolism at 48 h of imbibition. From the above results, we think real-time seed vigor of middle seeds should be higher than that of top seeds at 24 h of imbibition, but lower at 48 h of imbibition.
Plant Mol Biol Rep
Fig. 5 Enzymes activities in middle and top seeds imbibed for different hours. a Activity of alcohol dehydrogenase (ADH). b Activity of alphaamylase. c Activity of catalase (CAT). d Activity of peroxidase (POD).
Data represent the means ± SD of three replicates, with 30 seeds each. An ANOVA test followed by LSD post hoc test was performed. Single asterisk (*) is used for indicating significant difference (P < 0.05)
Aquaporin TIP3.1 and TIP3.2 Should Be Biomarkers of Seed Vigor
the embryonic axis cells (Bewley et al. 2013). A higher water uptake growth rate implied that more reserves within the embryonic axis cells of middle seeds were mobilized during phase II of water absorption. And TIP3.1 and TIP3.2, especially the latter, should trigger this process. In addition, water contents of top seeds were significantly higher than those of middle seeds at the same imbibition time (Fig. 3b). While, the levels of some important metabolisms in top seeds were lower than those in middle seeds at 24 h of imbibition (described above). The results indicated that although more water entered into top seeds, the activation of metabolisms was less efficient. That less water was transported into PSVs by less TIP3.1 and TIP3.2 in top seeds may be the key factor. Taken together, the results revealed that TIP3.1 and TIP3.2, especially the latter, should be useful biomarkers of seed vigor, which are valuable to characterize the complicated trait.
Generally, TIP3s are considered as marker proteins located at PSV membranes (Hunter et al. 2007; Jauh et al. 1999), which are abundant in dry seeds and seeds during early imbibition and then vanish when vacuoles recovering from PSVs (Novikova et al. 2014). With water inflow into PSVs during imbibition, the enzymes involved in protein mobilization are activated (Bewley et al. 2013). In this study, two aquaporins were differentially accumulated, GRMZM2G037327_P01 (aquaporin TIP3.1) and GRMZM2G103983_P01 (aquaporin TIP3.2). Both of them were highly accumulated in middle seeds at 24 h of imbibition. And TIP3.2 showed the maximum difference between the middle and top seeds in this study, 5.94 times higher in the former. These results suggested that more water may enter into PSVs in middle seeds compared with those in top seeds, and then more storage proteins were degraded in the middle seeds at 24 h of imbibition. This was indirectly confirmed by the higher water uptake growth rate of middle seeds during phase II of seed water absorption (Fig. 3c). Bewley et al. showed that further water uptake during phase II must be due to lowering water potential by degradation of polymeric reserves within
Middle Seeds May Suffer Less Fungal Damages Among all the DAPs between middle seeds and top seeds, the number of Disease/Defense class proteins was the largest, accounting for 34.9%, 29.3%, and 18.8% of DAPs at 0 h, 24 h, and 48 h of imbibition, respectively. This indicated that
Plant Mol Biol Rep
the resistance of middle and top seeds towards stress conditions differed. Among Disease/Defense class DAPs, heat shock proteins were the most abundant, including not only small molecular weight heat shock proteins, such as GRMZM2G437100_P01 (16.9 kDa class I heat shock protein 2), GRMZM2G331701_P01 (22.0 kDa heat shock protein), GRMZM5G803365_P01 (26.5 kDa heat shock protein), and GRMZM2G149647_P01 (small heat shock protein, chloroplastic), but also high molecular weight heat shock proteins, such as GRMZM2G112165_P01 (heat shock protein 81-1) and GRMZM5G833699_P01 (heat shock protein 90-1). Interestingly, all the heat shock proteins except for GRMZM2G112165_P01 (heat shock protein 811) showed lower accumulation in the middle seeds compared with top seeds (Online Resources 2–4). Catusse et al. (2011) reported the positive relation between high-vigor seeds and increased accumulation level of small molecule heat shock proteins. However, in this study, fewer small molecule heatshock proteins accumulated in the high-vigor middle seeds than in the top seeds at the three time points, indicating that the accumulation of small molecule heat shock proteins is not a universal index of seed vigor. Fungal infection can affect seed germination and seedling growth (Kirkpatrick and Bazzaz 1979). In the present study, several fungal infection response proteins, such as chitinase A1 (GRMZM2G051943_P01), glucan endo-1,3-beta-glucosidase GI (GRMZM2G125032_P01), and two pathogenesis-related proteins (AC205274.3_FGP001 and GRMZM2G402631_P01), showed lower accumulation in the middle seeds, suggesting that the top seeds might suffer more fungal infection. During seeds drying, membranes’ integrity is lost (Simon 1974). When seeds are imbibed, solutes such as sugars, amino acids, and proteins are leaked from the cytoplasm (Bewley et al. 2013). The nutrients feed some pathogens, which then attack seeds, and even lead to pre-emergence mortality (Simon 1974). In this study, the leachate conductivity in the top seeds was significantly higher than that in the middle seeds (Fig. 3a), indicating that more nutrients in the top seeds had leaked into the imbibition solution during imbibition. This contributed to the propagation and growth of the fungi and could have caused the fungi to attack the top seeds more than the middle seeds. In addition, fungi can attack plants through the secretion of various hydrolases, including xylanase, to degrade plant cell walls, whereas plants inhibit the enzyme activity by synthesizing the corresponding inhibitors (Misas-Villamil and Van der Hoorn 2008), thus resisting the attacks of fungi to cell wall. In this study, we also found that the accumulation of xylanase inhibitor protein 1 (GRMZM2G162359_P01) in the middle seeds was higher than that in the top seeds at 24 h of imbibition (Online Resources 3), indicating that the middle seeds of the maize ear were more resistant against fungal attacks to cell wall. All the results suggested that middle seeds may suffer less fungal damages during imbibition.
Conclusions In this study, the differences of proteomic and some physiological profiles of seeds from middle and top sections of maize ears were investigated. Results showed that a total of 159 DAPs were identified between seeds from the two sections at 0 h, 24 h, and 48 h of imbibition. The DAPs belonged to various functional classes, such as Disease/Defense, Metabolism, Energy, Protein Destination and Storage, and Protein Synthesis, indicating these cellular processes were different between the middle seeds and top seeds at the three time points of imbibition. DAPs involved in protein synthesis and energy production, positively related to seed vigor, exhibited higher accumulation in middle seeds at 24 h of imbibition, but lower accumulation at 48 h of imbibition, indicating that realtime seed vigor of middle seeds should be higher than that of top seeds at 24 h of imbibition, but lower at 48 h of imbibition. TIP3.1 and 3.2, which mediate water inflow into protein storage vacuoles, then activating enzymes involved in reserve mobilization, showed higher accumulation in middle seeds at 24 h of imbibition. Together with the higher water uptake growth rate of middle seeds during phase II of seed water absorption, we think TIP3.1 and TIP3.2, especially the latter, should be useful biomarkers of seed vigor. In addition, our data also suggested that the middle seeds may suffer less fungal damages during imbibition. Our work contributes to understand how seed vigor is influenced by growth position in plant. Acknowledgements We thank Professor Gerhard Leubner, Royal Holloway, University of London, UK, for reading and providing valuable comments on this manuscript. Author Contributions YL and CZ planned and designed the research. HQ, PZ, and KS conducted experiments. HQ, YL, and CZ analyzed the data. YL wrote the paper. Funding Information This work was supported by the grants from the National Natural Science Foundation of China (31271808 and 31771890) and the National Key Research and Development Program of China (2018YFD0100901).
Compliance with Ethical Standards Conflict of Interest The authors declare that they no conflict of interest.
References Bernfeld P (1955) Amylases α and β. Method Enzymol 1:149 Bevan M, Bancroft I, Bent E et al (1998) Analysis of 1.9 Mb of contiguous sequence from chromosome 4 of Arabidopsis thaliana. Nature 391:485 Bewley JD, Bradford KJ, Hilhorst HW, Nonogaki H (2013) Seeds, 3rd edn. Springer, New York Campo S, Carrascal M, Coca M, Abián J, San Segundo B (2004) The defense response of germinating maize embryos against
Plant Mol Biol Rep fungal infection: a proteomics approach. Proteomics 4:383– 396 Catusse J, Meinhard J, Job C, Strub J-M, Fischer U, Pestsova E, Westhoff P, van Dorsselaer A, Job D (2011) Proteomics reveals potential biomarkers of seed vigor in sugarbeet. Proteomics 11:1569–1580 Chu P, Chen H, Zhou Y, Li Y, Ding Y, Jiang L, Tsang EWT, Wu K, Huang S (2012) Proteomic and functional analyses of Nelumbo nucifera annexins involved in seed thermotolerance and germination vigor. Planta 235:1271–1288 Cordero M, Raventos D, San Segundo B (1994) Differential expression and induction of chitinases and β-1, 3-glucanases in response to fungal infection during germination of maize seeds. Mol Plant Microbe Interact 7:23–31 Finch-Savage W, Bassel G (2016) Seed vigour and crop establishment: extending performance beyond adaptation. J Exp Bot 67:567–591 Gattolin S, Sorieul M, Frigerio L (2011) Mapping of tonoplast intrinsic proteins in maturing and germinating Arabidopsis seeds reveals dual localization of embryonic TIPs to the tonoplast and plasma membrane. Mol Plant 4:180–189 Gutterman Y (2000) Maternal effects on seeds during development. In: Fenner M (ed) Seeds: the ecology of regeneration in plant communities, 2nd edn. CABI publishing, London, pp 59–84 Hampton JG, Tekrony DM (1995) Handbook of vigour test methods. The International Seed Testing Association, Zurich Hanson AD, Jacobsen JV (1984) Control of lactate dehydrogenase, lactate glycolysis, and α-amylase by O2 deficit in barley aleurone layers. Plant Physiol 75:566–572 Hunter PR, Craddock CP, Di Benedetto S, Roberts LM, Frigerio L (2007) Fluorescent reporter proteins for the tonoplast and the vacuolar lumen identify a single vacuolar compartment in Arabidopsis cells. Plant Physiol 145:1371–1382 Imai A, Komura M, Kawano E, Kuwashiro Y, Takahashi T (2008) A semi-dominant mutation in the ribosomal protein L10 gene suppresses the dwarf phenotype of the acl5 mutant in Arabidopsis thaliana. Plant J 56:881–890 Jauh G-Y, Phillips TE, Rogers JC (1999) Tonoplast intrinsic protein isoforms as markers for vacuolar functions. Plant Cell 11:1867–1882 Kato M, Shimizu S (1987) Chlorophyll metabolism in higher plants. VII. Chlorophyll degradation in senescening tobacco leaves: phenolicdependent peroxidative degradation. Can J Bot 65:729–735 Kiesselbach TA (1999) The structure and reproduction of corn, 50th Anniversary Edition. Cold Spring Harbor Laboratory Press, New York Kirkpatrick B, Bazzaz F (1979) Influence of certain fungi on seed germination and seedling survival of four colonizing annuals. J Appl Ecol 16:515–527 Lamikanra O, Watson MA (2001) Effects of ascorbic acid on peroxidase and polyphenoloxidase activities in fresh-cut cantaloupe melon. J Food Sci 66:1283–1286 Li W, Zhao F, Fang W et al (2015) Identification of early salt stress responsive proteins in seedling roots of upland cotton (Gossypium hirsutum L.) employing iTRAQ-based proteomic technique. Front Plant Sci 6:732 Logan DC, Millar AH, Sweetlove LJ, Hill SA, Leaver CJ (2001) Mitochondrial biogenesis during germination in maize embryos. Plant Physiol 125:662–672 Maurel C, Verdoucq L, Luu D-T, Santoni V (2008) Plant aquaporins: membrane channels with multiple integrated functions. Annu Rev Plant Biol 59:595–624
Melroy DL, Herman EM (1991) TIP, an integral membrane protein of the protein-storage vacuoles of the soybean cotyledon undergoes developmentally regulated membrane accumulation and removal. Planta 184:113–122 Merchante C, Stepanova AN, Alonso JM (2017) Translation regulation in plants: an interesting past, an exciting present and a promising future. Plant J 90:628–653 Misas-Villamil JC, Van der Hoorn RA (2008) Enzyme-inhibitor interactions at the plant-pathogen interface. Curr Opin Plant Biol 11:380–388 Mondo VHV, Cicero SM (2005) Using image analysis to evaluate the quality of maize seeds located in different positions on the ear. Rev Bras Sementes 27:9–18 Novikova GV, Tournaire-Roux C, Sinkevich IA, Lityagina SV, Maurel C, Obroucheva N (2014) Vacuolar biogenesis and aquaporin expression at early germination of broad bean seeds. Plant Physiol Biochem 82:123–132 Obroucheva N, Sin’kevich I (2010) Aquaporins and cell growth. Russ J Plant Physiol 57:153–165 Postaire O, Verdoucq L, Maurel C (2007) Aquaporins in plants: from molecular structure to integrated functions. Adv Bot Res 46:75–136 Shen S, Zhang L, Liang X-G, Zhao X, Lin S, Qu LH, Liu YP, Gao Z, Ruan YL, Zhou SL (2018) Delayed pollination and low availability of assimilates are major factors causing maize kernel abortion. J Exp Bot 69:1599–1613 Shi Z, Fujii K, Kovary KM, Genuth NR, Röst HL, Teruel MN, Barna M (2017) Heterogeneous ribosomes preferentially translate distinct subpools of mRNAs genome-wide. Mol Cell 67:71–83 Simon E (1974) Phospholipids and plant membrane permeability. New Phytol 73:377–420 Soós V, Sebestyén E, Juhász A, Light ME, Kohout L, Szalai G, Tandori J, van Staden J, Balázs E (2010) Transcriptome analysis of germinating maize kernels exposed to smoke-water and the active compound KAR1. BMC Plant Biol 10:236 Sung TY, Delouche JC (1962) Relation of specific gravity to vigor and viability in rice seed. Proc Assoc Off Seed Anal 52:162–168 Turc O, Bouteillé M, Fuad-Hassan A, Welcker C, Tardieu F (2016) The growth of vegetative and reproductive structures (leaves and silks) respond similarly to hydraulic cues in maize. New Phytol 212:377–388 Tyerman S, Bohnert H, Maurel C, Steudle E, Smith J (1999) Plant aquaporins: their molecular biology, biophysics and significance for plant water relations. J Exp Bot 50:1055–1071 Wang W-Q, Ye J-Q, Rogowska-Wrzesinska A, Wojdyla KI, Jensen ON, Møller IM, Song S-Q (2013) Proteomic comparison between maturation drying and prematurely imposed drying of Zea mays seeds reveals a potential role of maturation drying in preparing proteins for seed germination, seedling vigor, and pathogen resistance. J Proteome Res 13:606–626 Wang W-Q, Liu S-J, Song S-Q, Møller IM (2015) Proteomics of seed development, desiccation tolerance, germination and vigor. Plant Physiol Biochem 86:1–15 Wu X, Liu H, Wang W, Chen S, Hu X, Li C (2011) Proteomic analysis of seed viability in maize. Acta Physiol Plant 33:181–191 Xin X, Lin XH, Zhou YC, Chen XL, Liu X, Lu XX (2011) Proteome analysis of maize seeds: the effect of artificial ageing. Physiol Plant 143:126–138
