Proteins were extracted from the various true seed samples (freshly harvested, DI water treated for 2 days, 5 mM citrate buffer (pH 2.0) treated for 2 days, and 5 mM citrate buffer (pH 7.0) treated for 2 days) and analyzed by 2-DE as described in Section 2.2.4. As shown in Figures 13 (embryos) and 14 (testae), there were some protein spots with significant changes (of at least ten-fold) which were obtained by comparing protein patterns of fresh seeds and treated seeds, and 45 and 70 dramatic changes of embryos and testae in abundances were observed, respectively. These protein spots were summarized in TABLE II. A total of 115 interesting spot proteins were excised from preparative 2-DE gels. After trypsin in-gel digestion, the proteins were analyzed by MALDI-TOF-MS or ESI-MS/MS. Among the changed proteins, three spots showed significant matches. MS data including peptide masses and identified amino acid sequences for spots of interest were summarized in TABLE III. Protein No. 8, 21, and 25 were identified as Prunus serotina (R)-mandelonitrile lyase 1 precursor, Vitis vinifera hypothetical protein, and Prunus dulcis prunin, respectively. It seemed important to discuss the functions of the identified proteins and related metabolic pathways involved in dormancy-breaking.
19
4. Discussion
In the germination experiments (Figure 2), low germination percentages of freshly harvested intact seeds of P. campanulata were observed, and most of seeds did not germinate under either warm or cold stratification, but warm plus cold stratification increased seed germination significantly. It indicated that a combination of warm and cold stratification is necessary for releasing dormancy completely. According to the previous researches (Chen et al., 2007), cold stratification alone stimulated a high percentage of the seeds to germinate, up to 70% for 8 weeks treatments, and it has been widely used for breaking seed dormancy and promoting the maximum percentage and rate of germination (International Seed Testing Association, 1999; Schopmeyer, 1974). However, our results (Figure 2) showed that small amounts of seeds germinated after cold stratification, and the germination percentage was only 18%. The differences in the germination of seeds in response to stratification were linked to fruit maturity which depended upon the year and the location of the seed harvested (Chen et al., 2007). Nevertheless, we could confirm that the seeds were alive for subsequent experiments because their germination percentages reached 100% through warm stratification for 6 weeks plus cold stratification for 8 weeks.
In this research, the true seeds which still kept their dormancy characteristics (Chen et al., 2007) were used as the experimental materials for studying the mechanism of P.
campanulata seed dormancy. With red rice and several other species, embryo pH decreased during dormancy-breaking process and subsequent germination (Footitt and Cohn, 1992). In true seeds of P. campanulata, pH values of testae and embryos declined as dormancy-breaking and germination were also found (Figure 3). The pH change of embryos was more rapid than it of testae. In 1992, Footitt and Cohn mentioned that multicellular systems of the plant and animal kingdoms seem to be a decrease in internal pH upon activation. Our results also presented that the tissues, testae and embryos, acidification was a prerequisite for the termination of dormancy.
20
We applied different pH environments with citrate buffer to induce the dormancy-breaking of the seeds. Figure 5 indicated that true seeds which were incubated with low pH buffers germinated faster and reached higher germination percentages than those in near neutral pH conditions. The radical growth of the true seeds was further examined to understand pH effects on other plant physiological phenomenon based on the results of the pH effects on dormancy-breaking and germination. As shown in Figure 6, the radicles of true seeds favored to grow in low pH environments instead of near neutral ones. Previous studies on seed dormancy of red rice showed that dormancy-breaking activity was pH-dependent. Incubation medium pH values that favored formation of the protonated species resulted in the highest germination percentages (Cohn et al., 1987).
Consequently, low pH treatments effectively promote seed dormancy-breaking and induce its radical growth.
We also used different type of the seeds, isolated embryos, to identify the pH effects on P. campanulata seeds. As shown in Figure 8, the seed radicles favored to grow in weak acidic environments, and there were similar characteristics in true seeds. It should be noted that pH 4.0 was the best condition for radical growth of isolated embryos, possibly resulting from the pH effects on ABA movement according to the previous studies (Kaiser and Hartung, 1981). In this situation (pH < pKa), un-dissociated species would be the major forms, and the main penetrating species between the cells and free space has been proven as un-dissociated ABA (Kaiser and Hartung, 1981). The pKa which was calculated as described in Section 3.2.2.2 was 4.4, and it was considerably closed to the theoretical value of ABA (pKa=4.8) as shown in Figure 8 (inset). Therefore, the seed radicles grew well in low pH values (pH≦4.0) because the un-dissociated ABA was easy to penetrate the tissues and move outside. But, poor radical growth of isolated embryos in acidic conditions, pH 2.0 and 3.0, was observed, and we considered that isolated embryos lacked testae protections resulted in this phenomenon. The radical growth of isolated embryos
21
(Figure 7) which was more sensitive to high concentration of the buffers than true seeds (Figure 4), and this may be due to isolated embryos lacked the protections of testae.
Figures 10 and 11 showed the effects of exogenous ABA under various pH conditions on the progress of dormancy-breaking and germination through the observation of seed germination and radical growth. Incubation in the presence of exogenous ABA obviously decreased the germination percentage (Figure 10) and radical length (Figure 11) of the true seeds at low pH values (from pH 2.0 to 4.0). The low pH of the incubation medium might produce a relevant effect on the accumulation of exogenous ABA. At the more acidic pH, the greater total amount of exogenous ABA entered the seed and favored to accumulate into the embryo and endosperm even above the levels of the untreated control in red rice (Gianinetti and Vernieri, 2007). By contrast, the ability of ABA to suppress germination and radical growth was strongly reduced at near neutral pH values.
Indeed, weak acids (like ABA) have low ability to penetrate through the seed coats at pH values higher than their pKa because the movement of the un-dissociated form of weak acids into seeds is favored (Cohn et al., 1987). In ABA quantification experiments (Figure 12), we found the difference between dormant and dormancy-breaking seeds which incubated with DI water for 2 and 10 days, respectively. There were similar ABA concentrations in dormant seeds even through DI water or different pH buffer treatments.
Thus, ABA should not be the key modulator and there might be other regulatory factors involved in pH effects on the dormancy-breaking of true seeds.
Studies of plant proteomic analyses used so far have been reported (Lee et al., 2006;
Cánovas et al., 2004). Proteomic tools offer the ways to analyze net-works of proteins that control important physiological reactions involved in seed dormancy-breaking/germination.
As results, the pH treatments effectively affected the dormancy-breaking of P.
campanulata true seeds, and the change in proteins following this treatment might be important factors in seed dormancy. According to the result as shown in Figure 5, true
22
seeds were treated for 2 days to study the pH effects on the process of the seed dormancy-breaking. The labeled protein spots identified by MS were listed in TABLE III.
In embryos, three proteins which exhibited dramatic changes after DI water or different pH buffer treatments were identified as (R)-mandelonitrile lyase 1 precursor and prunin which belonged to the genus Prunus except hypothetical protein (Garcia-Mas et al., 1995).
Amygdalin is hydrolyzed and generated hydrogen cyanide gas (HCN) and benzaldehyde by several enzymes including (R)-mandelonitrile lyase (MDL) (Zhou et al., 2002). Prunus species (Rosaceae) are a rich source of the cyanogenic diglucoside (R)-amygdalin [the β-gentiobioside of (R)-mandelonitrile] and its catabolic enzymes which accumulate in leaves and immature fruits (Zheng and Poulton, 1995). Also, changes in proteins probably indicate biochemical activities of the seed after the uptake of water (Lee et al., 2006). Here, (R)-mandelonitrile lyase 1 precursor exhibited dramatic decrease after DI water treatment because the dormancy of true seeds might be broken.
In embryos, the other identified proteins were Vitis vinifera hypothetical protein whose function was unknown at present (Velasco et al., 2007), and Prunus dulcis prunin, was known as globulin of the genus Prunus, which comprise the main family of storage proteins synthesized in seeds during embryogenesis (Garcia-Mas et al., 1995). The protein which was identified as prunin exhibited dramatic changes after the treatment at pH 2.0 that accelerated the dormancy-breaking of true seeds. Previous articles indicated that the fragmentation of prunin may possibly be used as an index of seed germination (Lee et al., 2006), and the GA contents of P. campanulata seeds increased during dormancy-breaking process (Chen et al., 2007). In other words, degradation of prunin which occurred during dormancy-breaking is probably related to GA induction. It will be the important evidences to study the effect of the degradation of prunin during the breaking of seed dormancy.
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5. Conclusion
P. campanulata seeds were suitable experimental materials for studying the mechanism of dormancy-breaking resulted from their dormancy characteristics. We confirmed that low pH treatments effectively promoted seed dormancy-breaking and induce radical growth. Results of our analysis provided evidences of the involvement of MDL and prunin in response to the dormancy-breaking of true seeds. The study of the pH effects on the proteomes of tree seeds will contribute to an understanding of the molecular basis of seed dormancy.
TABLE I
The pH values of citrate buffer upon addition of various ABAa concentrations
ABA concentrationb (μM)
5 mM citrate buffer
pH 2.0 pH 3.0 pH 4.0 pH 5.0 pH 6.0 pH 7.0
－ 2.12±0.015 3.06±0.007 4.01±0.011 5.05±0.049 6.04±0.026 7.09±0.014
1 2.13±0.010 3.06±0.020 4.01±0.035 5.08±0.030 6.07±0 7.00±0.042
10 2.14±0.014 3.05±0.023 4.02±0.021 5.06±0.056 6.06±0.020 7.00±0.015
100 2.11±0.017 3.03±0.025 4.00±0.026 5.01±0.011 6.01±0.015 6.99±0.005
aThe pH value of each solution was measured immediately (to avoid alkalinization by uptake of atmospheric carbon dioxide) with a normal pH electrode. And the pH value was recorded when the electrode registered a stable pH (<0.01 units min-1); this was generally achieved between 3 and 5 min. Values were means ± SD of three repeats independently.
bABA was dissolved in methanol whose final concentration was 0.1% (v/v).
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TABLE II
Changes in P. campanulata true seed proteins following various treatmentsa
Treatments
Increased protein spots numbers Decreased protein spots numbers
Embryo Testa Embryo Testa
DI water 4, 6, 9, 12, 14, 15, 16, 20, 21
47, 50, 52, 55, 57, 63, 66, 68, 69, 72, 75, 76, 77
3, 8, 10, 11, 13, 17, 18, 19 46, 48, 49, 51, 53, 54, 56, 58, 59, 60, 61, 62, 64, 65, 67, 70, 71, 73, 74 pH 2.0 24, 28, 29, 31, 32, 33, 34 47, 82, 86, 89, 92, 94 23, 25, 26, 27, 30, 35, 36 53, 58, 78, 79, 80, 81, 83, 84, 85, 87,
88, 91, 93, 95, 96, 97 pH 7.0 1, 37, 45 100, 101, 103, 106, 108, 109,
110, 111, 112, 113, 114
2, 5, 7, 19, 22, 38, 39, 40, 41, 42, 43, 44
48, 78, 81, 90, 98, 99, 102, 104, 105, 107, 115
aSpot changes were obtained by comparing protein patterns of fresh seeds and treated seeds (DI water, 5 mM citrate buffer (pH 2.0), and 5 mM citrate buffer (pH 7.0) for 2 days).
25
26
TABLE III
Identified proteins of P. campanulata true seedsa
Spot No. Protein name Identified sequence
Protein MW (kDa)/pI
Accession No. Species Experimental Theoretical
116/5.56 61.2/5.45 P52706 Prunus serotina (Zheng and Poulton, 1995)
21 Hypothetical protein MLVPHTIPICAAWSDDLMKK LVDNK DGLWQICNQV SAIME
34.0/4.80 15.1/4.12 CAN64396 Vitis vinifera
(Velasco et al., 2007)
25 Prunin GN LDFVQPPRADI FSPR 63.0/5.33 63.0/6.59 CAA55009 Prunus dulcis
(Garcia-Mas et al., 1995)
aProtein spots labeled with the same number in different gels indicate the same protein by analysis of ImageMaster 2D elite software. Except for spot No. 8, 21, and 25, other spots showed no good matches. Spot No. 8 and 21 were identified by MALDI-TOF-MS; spots No. 25 was identified by ESI-MS/MS.
hilum endocarp seed coat
cotyledons epicotyl hypocotyl
endosperm radicle
embryo
testa
Figure 1: Longitudinal section through a seed of P. campanulata. A small amount of endosperm adheres to the hypocotyl and radicle (modifying from Chen et al., 2007).
27
Stratification condition
Fresh Warm 6 weeks Cold 8 weeks Mixed 14 weeks
Germination percentage (%)
0 20 40 60 80 100
a ab
b
c
Figure 2: Germination percentage of intact P. campanulata seeds following warm or/and cold stratification. The period of each germination test was 12 weeks. The
“Warm 6 weeks” indicates that seeds were stratified at 30/20℃ for 6 weeks, and those stratified at 4℃ for 8 weeks are termed as “Cold 8 weeks”. The “Mixed 14 weeks”
indicates that the seeds were stratified at 30/20℃ for 6 weeks followed by 4 for 8 ℃ weeks. Columns with the same letter were not significantly different (P≦0.05).
28
Incubation (days)
Figure 3: Tissue pHs and germination percentage of P. campanulata true seeds during incubation at 30/20℃ for 20 days. The treated true seeds were separated into two parts, testae (filled circles) and embryos (open circles), which were homogenized and the pH value of each extract was measured immediately with a normal pH electrode. The germination (filled squares) was recorded every two days, and results were expressed as percent germination.
29
Citrate buffer concentration (mM)
DI water 1 5 10 20 50 100
Radicle length (mm)
0 2 4 6 8 10
pH=2.0 pH=3.0
Figure 4: Determination of optimal buffer concentration for the radical growth of true seeds. The true seeds were incubated with the citrate buffer in different concentrations at pH 2.0 (black columns) and 3.0 (gray columns), and DI water was used as control medium. The radical lengths of true seeds were recorded at 10 days after sowing (DAS).
30
Incubation (days)
0 5 10 15 20
Germination percentage (%)
0 20 40 60 80 100
DI water pH=2.0 pH=3.0 pH=4.0 pH=5.0 pH=6.0 pH=7.0
Figure 5: Germination of true seeds responded to the citrate buffer with different pHs. The true seeds were treated with the citrate buffer (5 mM) with variant pHs, and DI water (filled circles) was used as control medium. Germination, judged by radical protrusion of at least 2 mm, was recorded every two days.
31
Incubation condition
DI water pH 2.0 pH 3.0 pH 4.0 pH 5.0 pH 6.0 pH 7.0
Radicle length (mm)
0 2 4 6 8
Incubation condition
DI water pH 2.0 pH 3.0 pH 4.0 pH 5.0 pH 6.0 pH 7.0
Germination percentage (%)
0 20 40 60 80
Figure 6: Effects of different pH conditions on germination and radical growth of true seeds. The treated media were the citrate buffer (5 mM) with various pHs (from pH 2.0 to 7.0), and DI water was used as control medium. The radical lengths and germination percentages (inset) of true seeds were recorded at the 10th day after sowing.
32
Citrate buffer concentration (mM)
DI water 1 5 10 25 50
Radicle length (mm)
0 2 4 6 8
pH=4.0 pH=7.0
Figure 7: Determination of optimal buffer concentration for the radical growth of isolated embryos. The isolated embryos were incubated with the citrate buffer in various concentrations at pH 4.0 (black column) and 7.0 (gray column), and DI water was used as control medium. The radical lengths of isolated embryos were collected at 5 DAS.
33
Incubation conditions
Figure 8: Effects of different pH conditions on radical growth of isolated embryos. The treated media were the citrate buffer (5 mM) with various pHs (from pH 2.0 to 7.0), and DI water was used as control medium. The radical lengths of isolated embryos were recorded at 5 DAS by tweezers and section paper. Columns with the same letter were not significantly different (P≦0.05). The pKa (inset) was calculated using nonlinear regression with SigmaPlot 2001, Version 7.0 and Enzyme Kinetics Module, Version 1.1 (SPSS Inc., Chicago, IL).
34
ABA concentration (μM)
Buffer Buffer+Methanol 0.1 1 10 100
Radicle length (mm)
0.0 0.5 1.0 1.5 2.0
2.5 *
Figure 9: Radical growth of true seeds responded to different concentrations of exogenous ABA. The true seeds were incubated with 5 mM citrate buffer (pH=5.0) containing different concentrations of ABA which dissolved in 0.1% (v/v) methanol.
The 5 mM citrate buffer and citrate buffer with 0.1% methanol (v/v) were used as control solutions. The radical lengths of true seeds were recorded at the 10th day after sowing. The differences of the radical growth of true seeds were analyzed by t-test using SigmaPlot 2001, Version 7.0 (SPSS Inc., Chicago, IL), and the star symbol indicated P<0.05.
35
pH values of 5 mM citrate buffers
pH values of 5 mM citrate buffers
DI water pH 2.0 pH 3.0 pH 4.0 pH 5.0 pH 6.0 pH 7.0
Figure 10: The exogenous ABA effects on germination of true seeds under the buffer treatments at different pHs. The seeds were treated with 5 mM citrate buffer from pH 2.0 to 7.0 either alone or in the presence of 1 μM ABA which were dissolved in 0.1% methanol (v/v). In this experiment, DI water was used as a control solution.
The germination percentages of true seeds were recorded at 10 DAS. The differences of the germination percentages between the seeds which incubated with (w/, gray column) and without (w/o, black column) ABA in the same pH environments were shown as inset.
36
pH values of 5 mM citrate buffers
pH values of 5 mM citrate buffers
DI water pH 2.0 pH 3.0 pH 4.0 pH 5.0 pH 6.0 pH 7.0
Figure 11: The exogenous ABA effects on radical growth of true seeds under the buffer treatments at different pHs. The seeds were treated by 5 mM citrate buffer from pH 2.0 to 7.0 either alone or in the presence of 1 μM ABA which were dissolved in 0.1% methanol (v/v). In this experiment, DI water was used as a control solution. The radical lengths of true seeds were recorded at 10 DAS. The differences of the radical length between the seeds which incubated with (w/, gray column) and without (w/o, black column) ABA in the same pH environments were shown as inset.
37
ABA concentrations (pg seed-1)
0 1000 2000 3000 4000
Treated conditions
pH 7.0 (2 days) pH 2.0 (2 days) DIW (10 days) DIW (2 days) Fresh
Figure 12: ABA contents of the fresh and treated true seeds. The seeds were incubated with either DI water for 2 and 10 days or citrate buffers (5 mM) for 2 days.
The ABA concentrations of different treated seeds were measured with GC-MS as described in Section 2.2.5. Results were presented as means ± SD from 2 replicates of 100 seeds each.
38
(A) (B)
Figure 13: Protein patterns of embryos of true seeds with different treatments.
The embryos of fresh (A, untreated), DI water treated (B), 5 mM citrate buffer (pH 2.0) treated (C), and 5 mM citrate buffer (pH 7.0) treated (D) true seeds were isolated for 2-D electrophoretic analysis. Labeled spots of the gels were those differentially expressed by at least ten-fold during the various treatments.
39
(A) (B)
Figure 14: Protein patterns of testae of true seeds with different treatments.
The testae of fresh (A, untreated), DI water treated (B), 5 mM citrate buffer (pH 2.0) treated (C), and 5 mM citrate buffer (pH 7.0) treated (D) true seeds were isolated for 2-D electrophoretic analysis. Labeled spots of the gels were those differentially expressed by at least ten-fold during the various treatments.
40
41
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