Evidence that cranberry juice may improve augmentationindex in overweight men Guillaume Ruel, Annie Lapointe, Sonia Pomerleau, Patrick Couture, Simone Lemieux,Benoît Lamarche, Charles Couillard Institute of Nutraceuticals and Functional Foods, Université Laval, Québec (Canada), G1V 0A6 The stiffening of arteries is a key step in atherogenesis leading to cardiovascular disease. It
Rtp035 1.9Journal of Plant Ecology Advance Access published January 28, 2010
Role of endogenous hormones, glumes, endosperm and doi: 10.1093/jpe/rtp035 temperature on germinationof Leymus chinensis (Poaceae)seeds during development Hongyuan Ma1,2, Zhengwei Liang1,2, *, Haitao Wu1, Lihua Huang1,2 and Zhichun Wang1,2 1 Northeast Institute of Geography and Agricultural Ecology, Chinese Academy of Sciences, Changchun 130012, People's Republic of China 2 Da'an Sodic Land Experiment Station of China, Da'an 131317, People's Republic of China*Correspondence address. Northeast Institute of Geography and Agricultural Ecology, Chinese Academy ofSciences, Changchun 130012, People's Republic of China. Tel: +86-431-85542347; Fax: +86-431-85542347;Email: [email protected] Important Findings Leymus chinensis is an original dominant plant in the Songnen grass- Of the four endogenous hormones monitored, only the concentra- land, and it has great value for restoration of severely degraded land.
tion of ZR differed significantly between the beginning and the However, seeds are dormant, and low germination percentage is end of seed development (increased); the GA3/ABA ratio also did a problem for restoring L.chinensis grassland. The mechanism of seed not differ. Rank of germination percentage of control at the three tem- dormancy is not been well understood. The primary aims of the pres- perature regimens was 5/28°C > 16/28°C > 5/35°C. Germination per- ent study were to investigate the dormancy mechanism of L.chinensis centage of the naked-half seeds reached 100% under the three seeds (caryopses) with reference to the role of embryo-covering temperature regimens. We concluded that dormancy of L.chinensis layers, endogenous hormones and temperature.
seeds is not mainly controlled by endogenous hormones. Germina-tion temperature, mechanical resistance of glumes and inhibition of endosperm are the main factors controlling dormancy and germina- Changes in concentration of the endogenous hormones GA3, indole- tion of L.chinensis seeds.
acetic acid (IAA), zeatin riboside (ZR) and abscisic acid (ABA) in L.chi-nensis seeds from anthesis to maturity were measured by the enzyme- Keywords: endogenous hormones d temperature d Leymus linked immunosorbent assay method. Germination at different stages chinensis d anthesis d seed d germination of maturity were tested at 16/28°C, 5/28°C and 5/35°C for intact seedswith glumes (control), intact seeds with glumes removed (naked- Received: 25 August 2009 Revised: 19 November 2009 Accepted: whole seeds) and intact seeds with glumes and one-half of the endo- sperm removed (naked-half seeds).
drolyze the endosperm and by increasing the growth potentialof the embryo. ABA mainly induces seed dormancy during The role of hormones in controlling seed dormancy is an active later phases of seed maturation (Debeaujon and Koornneef area of research. Some researchers considered that GA and absci- 2000). The balance of these two hormones controls seed dor- sic acid (ABA) are the main hormones controlling primary dor- mancy and germination (Hilhorst and Karssen 1992; Iglesias mancy. Endogenous GA is thought to control germination by and Babiano 1997; Koornneef et al. 2002). Indoleacetic acid inducing the expression of genes encoding enzymes that hy- (IAA) and the cytokinins zeatin (Z) and zeatin riboside (ZR) Ó The Author 2010. Published by Oxford University Press on behalf of the Institute of Botany, Chinese Academy of Sciences and the Botanical Society of China.
All rights reserved. For permissions, please email: journals.perm[email protected] Journal of Plant Ecology also play roles in breaking seed dormancy and promoting ger- tigated under three temperature regimens in this study. The mination. IAA decreases sensitivity to endogenous ABA (Mat- aims of the study were to further understand the mechanisms illa 2000) or inhibits the action of ABA (Beaudoin et al. 2000; of dormancy and germination in L.chinensis seeds and to pro- Ghassemian et al. 2000). The main effects of Z and ZR are to vide theoretical and technical information for improving seed increase the number of cells and therefore break seed dor- mancy (Yi et al. 1997). However, not all research has supportedsuch viewpoints. For example, Bewley (1997) reported that MATERIALS AND METHODS the contents of ABA in dormant and non-dormant seeds weresimilar.
The effect of seed coat on dormancy has been studied in In Songnen meadow grassland, anthesis in L.chinensis begins in many other species, and a number of mechanisms have been the end of May or beginning of June and seed setting occurs in advanced to explain the effect: (i) mechanical restriction on the middle of June. Seeds of L.chinensis from the onset of germination of the embryo, or the ‘mechanical barrier' model; anthesis to maturity were collected from the Da'an Sodic Land (ii) prevention of exit of inhibitors from the embryo, or the Experiment Station (45°36# N, 123°53# E), Jilin Province, ‘inhibitor exit' model; (iii) chemical inhibition of germination, China. Panicles with similar size and development stage were or the ‘seed coat inhibitor' model; (iv) restriction on water up- labeled on 28 May 2007. We collected 100 panicles on 29 May take, or the ‘water-impermeable' model and (v) restriction on (0 day after anthesis, i.e. DAA), 3 June (5 DAA), 8 June oxygen uptake, or the ‘oxygen diffusion model' (Bewley and (10 DAA), 13 June (15 DAA), 18 June (20 DAA), 23 June Black 1994; Morris and Tieu 2000).
(25 DAA), 28 June (30 DAA), 3 July (35 DAA) and 18 July Leymus chinensis (Trin.) Tzvel. is a rhizomatous perennial (50 DAA) 2007. This period spans the beginning of anthesis grass with high ecological and economic importance and to the completion of seed maturity. On each collection day, was originally a dominant species in the Songnen grassland 50 panicles were put into plastic bags and stored at in China. Over the past few years, L.chinensis grassland resto- for later measurement of hormones, and the other 50 were ration has attracted the attention of many government officials stored in paper bags at room temperature until germination and researchers. However, the low germination percentage tests were carried out.
(10–20%) of L.chinensis seeds has proved to be a stumblingblock to the restoration of grassland. It has prevented the at- Measurement of endogenous hormone tainment of expected ecological and economic goals for resto- Individual seeds of L.chinensis stored at 80°C were separated ration and has also led to substantial quantities of seed being by hand from the panicles. Weighed samples of seeds (gener- lost. Yi (1994) reported that germination of freshly matured ally 1 g fresh weight, i.e. FW) were added to 4 ml of extraction L.chinensis seeds was low under natural conditions, and the buffer consisting of 80% (v/v) methanol with 1 mM butylhy- mechanisms controlling seed dormancy have not been well droxytoluene. The samples were ground by mortar and pestle understood (Zhang et al. 2005). Earlier reports attributed the in an ice bath and then transferred to 10-ml tubes. The mortar dormancy of L.chinensis seeds mainly to ABA in caryopses, was washed three times after each sample with 6 ml of extrac- glumes and endosperm (Yi et al., 1993; Yi 1994; Yi et al.
tion buffer, which was then added to the tubes. Samples were 1997). They considered that the ratio of promoting hormones extracted at 4°C for 4 h and then centrifuged at 3500 r.p.m. for (GA3, IAA, Z, ZR) content to that of ABA played a critical role 10 min. The supernatant of each extract was passed through in seed germination, with high germination percentage a C18 solid-phase extraction column and then blown dry with obtained when the promoting hormones/ABA ratio was high.
N2. Changes of the plant endogenous GA3, ABA, ZR and IAA Ma et al. (2008c) reported that the lemma is on the same side of were quantified by enzyme-linked immunosorbent assays as the seed as the embryo, and they are tightly appressed to each described by Hess et al. (2002) and Zhang (2007). Three rep- other and the mechanical resistance of lemma was one of the licates were analyzed from each collection date.
most important factors in controlling the seed dormancy. Maet al. (2008a) also found that exogenous hormones had little effect on improving seed germination of L.chinensis.
The germination tests were carried out from August to Octo- The previous studies on seed dormancy of L.chinensis focused ber 2007. Germination was tested on controls (no pretreat- mainly on the role of endogenous hormones in mature seeds; fewer have examined whole seed development. The relation- pretreatments: (i) removing the glumes (naked-whole seeds) ships between hormone changes and germination of seeds and (ii) removing the glumes and then surgically removing with and/or without glumes (palea and lemma) and endo- half of the endosperm (naked-half seeds). Each treatment sperm under different temperature regimens have not been consisted of three replicates of 30 seeds each. Seeds were sur- reported. Therefore, hormone changes in L.chinensis seeds from face sterilized in 0.1% mercuric chloride for 10 min and then anthesis to maturity and germination of whole seeds (caryop- washed with distilled water several times. The seeds were ses) with and without glumes removed and of seeds with then sown in 9-cm-diameter Petri dishes lined with two glumes and one-half of the endosperm removed were inves- layers of filter paper saturated with 6 ml of distilled water.
Ma et al. Germination of Leymus chinensis (Poaceae) seeds The dishes were placed in an incubator for 20 days at 16/28°C (which simulates the late July temperature regimen in the Changes in concentration of GA research area), 5/28°C and 5/35°C (12 h/12 h, in which 3, IAA, ZR and ABA 5°C represents cold stratification temperature to break seed As shown in Fig. 1a, concentrations of endogenous GA3 de- dormancy, and 35°C represents conditions during hotter than creased from 0 to 10 DAA, reaching the minimum value of average summer). The light sources consisted of a mixture of 886.05 ng(gFW) 1 at 10 DAA, and then gradually increasing fluorescent and incandescent light (54 lmol m 2s 1, 400–700 to a maximum of 5378.9 ng(gFW) 1 at 50 DAA. However, nm), which was given during the high temperature period.
GA3 did not change significantly (P < 0.05) during the whole Water was added to the dishes when necessary to keep them development process except of that at 10 DAA.
continuously moist. The dishes were distributed randomly in In Fig. 1b, endogenous IAA concentration basically showed the incubator and their positions changed daily. A seed was a steady and insignificant (P > 0.05) decrease between 0 and 10 considered to have germinated when the radical protruded DAA, after which it increased from 983.1 ng(gFW) 1 at 10 through the glumes (for the control seeds) or seed coat DAA to 3376.8 ng(gFW) 1 for 35 DAA (P < 0.05). Then, (for the naked seeds). The number of germinated seeds IAA decreased to 2610.4 ng(gFW) 1 for 50 DAA, which was counted daily or ;5 days over the 20-day incubation was not significantly (P < 0.05) different from the concentra- period. Mean germination time (MGT) was calculated as tions from 15 to 35 DAA.
described by Duan et al. (2004).
As shown in Fig. 1c, endogenous ZR concentration remained low (446.5–581.5 ng(gFW) 1) from 0 to 10 DAA Statistical analyses and no significant differences were observed between them Germination percentages were arcsine transformed before sta- (P > 0.05). ZR concentration then increased significantly to tistical analysis, and means were compared by two-way anal- 1863.3 ng(gFW) 1 by 15 DAA. It increased slowly and ysis of variance (ANOVA) and protected least significant steadily (1863.3–3137.1 ng(gFW) 1) between DAA 15 and difference test (P = 0.05). Duncan's test was used for multiple 50, but the difference was not significant.
comparisons among all the treatments. Analyses were carried As shown in Fig. 1d, endogenous ABA concentration out using SPSS, version 13.0.
remained low (5272.8–6581.4 ng(gFW) 1) from 0 to 10 Figure 1: changes in endogenous hormone concentrations in Leymus chinensis seeds during development. Bars are 6SE, and values with differentlowercase letters are significantly different at the 0.05 level.
Journal of Plant Ecology DAA (P > 0.05) and increased significantly (P < 0.05) to a steady significant increase in germination was found from 12 478.3 ng(gFW) 1 by 35 DAA (P < 0.05). However, by 2.2% (15 DAA) to 91.1% (30 DAA), with no further significant 50 DAA, ABA concentration had decreased to 6294.9 increase after 30 DAA. Rank order of germination percentages ng(gFW) 1, which differed significantly only from that at of seeds harvested at 15–20 DAA was control > naked-half = 35 DAA (P < 0.05).
naked-whole (Fig. 3a and b); at 25 DAA, control = naked-half> naked-whole (Fig. 3c) and at 30–50 DAA, naked-half > con- Changes in ratio of promoting/inhibitory hormones trol = naked-half (Fig. 3d–f).
Ratios of GA3/ABA, ZR/ABA and IAA/ABA were <1.0 duringseed development, while the ratio of (GA Germination at 5/28°C ABA was >1 at 0 and 50 DAA, i.e. 1.16 and 1.77, respectively As shown in Fig. 4, with the increase of seed maturity, ger- (Fig. 2). Ratios of GA3/ABA, IAA/ABA and (GA3 + ZR + ABA + mination of control seeds showed a significant increase be- IAA)/ABA showed a tendency to decrease from 0 to 10 DAA, tween 15 DAA (25.6%) and 20 DAA (83.3%), with no and minimum values of 0.13, 0.15, 0.37, respectively, oc- significant differences in germination observed in the seeds curred at 10 DAA.
from DAA >20. A steady significant increase was foundin the germination of naked-whole seeds from 15 DAA (0) Effects of glumes, endosperm and temperature on to 25 DAA (85.6%), with no significant increase after 25 DAA. For the naked-half seeds, there was a steady signif- Germination at 16/28°C icant increase from 0 (15 DAA) to 88.9% (30 DAA) and no As shown in Fig. 3, with the increase of seed maturity, germi- significant increase after 30 DAA. Rank order of germination nation of control seeds significantly increase from 26.7% (15 percentages after the 20-day incubation of seeds harvested DAA) to 63.3% (20 DAA), and while no significant differences at 15–20 DAA was control > naked-whole naked-half were observed between 20, 25, 35 and 50 DAA. A steady sig- (Fig. 4a and b); at 25 DAA, naked-half naked-whole > con- nificant increase in germination was found in the naked- trol (Fig. 4c) and at 30–50 DAA, naked-half > naked-whole > whole seeds from 15 to 50 DAA. For the naked-half seeds, control (Fig. 4d–f).
Figure 2: changes in ratio of promoting to inhibitory hormones in Leymus chinensis seeds during development. Bars are 6SE, and values withdifferent lowercase letters are significantly different at the 0.05 level.
Ma et al. Germination of Leymus chinensis (Poaceae) seeds Figure 3: germination at 16/28°C of Leymus chinensis seeds collected on 15 (a), 20 (b), 25 (c), 30 (d), 35 (e) and 50 (f) DAA. Germination per-centage values of the 20-day incubation time with different lowercase letters are significantly different at the 0.05 level.
Figure 4: germination at 5/28°C of Leymus chinensis seeds collected on 15 (a), 20 (b), 25 (c), 30 (d), 35 (e) and 50 (f) DAA. Germination percentagevalues of the 20-day incubation time with different lowercase letters are significantly different at the 0.05 level.
Germination at 5/35°C there was a steady significant increase from 24.4% (15 DAA) As shown in Fig. 5, there was a steady increase in the germina- to 97.8% (25 DAA), and no further significant increase follow- tion of control seeds during the maturity process from 15 DAA ing 25 DAA. Rank order of germination percentages after the (0) to 35 DAA (41.1%), with no significant difference in germi- 20-day incubation of seeds harvested at 15–20 DAA was control nation between 35 DAA and 50 DAA (42.2%). A steady signif- > naked-whole naked-half (Fig. 5a and b). No naked-whole icant increase in germination of naked-whole seeds occurred or naked-half seeds germinated at 15 DAA (Fig. 5a and b). At from 15 DAA (4.4%) to 30 DAA (97.8%), and no further sig- 25–50 DAA, no significant differences in germination were nificant increase following 30 DAA. For the naked-half seeds, found between the treatments of naked-whole and naked-half Journal of Plant Ecology Figure 5: germination at 5/35°C of Leymus chinensis seeds collected on 15 (a), 20 (b), 25 (c), 30 (d), 35 (e) and 50 (f) DAA. Germination percentagevalues of the 20-day incubation time with different lowercase letters are significantly different at the 0.05 level.
seeds. The rank order of germination percentages was naked-whole naked-half > control (Fig. 5c–f).
Rate of germination at three temperature regimens The rank order of MGT of L.chinensis seeds at the three temper-ature regimens were control > naked-whole > naked-half seedsat 20–50 DAA. The longest MGT, 17.4 days, was for the controlat 5/35°C, and the shortest, 3.0–3.1 days, for naked-half seeds atall three temperature regimens (Fig. 6). The two-way ANOVAshowed that DAA, temperature, treatments and their two-wayinteractions significantly (P < 0.01) affected MGT (Table 2). Theimportance of the three factors and their interactions on MGTwas treatment > temperature > maturity > temperature 3 treat-ment > maturity 3 temperature.
Correlation between hormone contents and seedgermination percentage As shown in Table 1, although concentrations of GA3, IAA andZR were positively correlated with germination percentage,the differences were not significant (P > 0.05) with the excep-tion of ZR, which was significantly correlated with germina-tion percentages of naked-whole (r = 0.995, P < 0.01) andnaked-half (r = 0.874, P < 0.05) seeds. There were also no sig-nificant correlations between the ABA content and the germi-nation of seeds with three different treatments.
Figure 6: MGT of Leymus chinensis seeds collected on 15, 20, 25, 30, 35 and 50 DAA germinated at 16/28°C, 5/28°C and 5/35°C.
Relationship between endogenous hormones andgermination percentage They reported that ABA content increased and GA3 decreased Yi (1994) and Yi et al. (1997) indicated that the reason for low throughout seed development (Yi et al. 1997). Zhang et al.
germination of L.chinensis seeds was the high content of ABA (2007) also reported that contents of all hormones were higher and low content of GA3 and IAA in glumes and endosperm.
in the early stages of development than in the late stages. Large Ma et al. Germination of Leymus chinensis (Poaceae) seeds Table 1: correlation coefficients between hormone contents and Table 2: effects of maturity, temperatures, treatment and their germination percentage (GP) of seeds in different developmental interaction on germination of Leymus chinensis seeds Naked-whole seeds DAA 3 DAA 3 Temp 3 temp 3 153.7** 46.6** 278.0** 79.8** 19.5** 30.1** 252.0** Values in the table are F values; **P < 0.01, ns: P > 0.05.
GP, germination percentage.
*P < 0.05; **P < 0.01; ns: P > 0.05.
MGP, mean germination percentage.
amounts of exogenous ABA (25–100 lgg 1) and GA3 (100–600lgg 1) and IAA (100–600 lgg 1) had no inhibitory or obvious germination for the same treatments after 30 DAA, especially promotional effects on seed germination of L.chinensis seeds (Ma for naked-half seeds, when dormancy could be completely re- et al. 2008a), which is consistent with the results of this study.
moved by some treatments on the seeds. However, these results However, Liu et al. (2004) showed that GA are inconsistent with those reported by Liu et al. (2004) that 3 significantly im- proved germination of isolated embryos of L.chinensis but not maximum germination of L.chinensis seeds occurred ;11 that of the intact seeds with glumes (palea and lemma). Zhang DAA and then decreased gradually from 11 to 15 DAA; no seeds et al. (2006) showed that 1000 lgg 1 GA <9 DAA or >16 DAA germinated. Isolated embryos 11–16 DAA 3 improved germina- tion of L.chinensis intact seeds from 10.0 to 42.3%. Our study germinated to high percentages, and germination percentages decreased significantly from 17 to 28 DAA (Liu et al. 2004).
3, IAA, ZR and ABA were not correlated with germination of intact seeds of L.chinensis. GA Field observations by the authors indicate that seeds germi- 3 content was high- est but changed only a little during the whole development pro- nate at any time from summer to early autumn, whenever tem- cess. Concentrations of IAA and ZR basically showed a tendency perature and soil moisture are suitable. Temperature determines to increase during development, while ABA increased during germination time by (i) influencing germination directly and (ii) the early development stages and then decreased from 35 to regulating dormancy (Bouwmeester and Karssen 1992; Bra¨ndel 50 DAA. Germination of L.chinensis seeds increased with in- 2004). Baskin and Baskin (1998) considered temperature to be crease in maturity to 30 DAA and then remained constant after the major environmental factor causing changes in dormancy 30 DAA, which showed that the degree of dormancy decreased states. It plays a critical role in germination and dormancy of with an increase of maturity during 0–30 DAA.
Haloxylon ammodendron (Huang et al. 2003), Drosera anglica From Table 1, it could be considered that ZR might be one of (Baskin et al. 2001), 14 wetland Carex species (Kettenring the main hormones promoting germination of the L.chinensis na- and Galatowitsch 2007), Halocnemum strobilaceum (Qu et al.
ked-half seeds after removing the glumes and part of endosperm.
2008) and L.chinensis (Ma et al. 2008b). Alternating tempera- Yi et al. (1997) reported that the effects of Z + ZR on the germi- tures usually are more favorable for germination than constant nation of L.chinensis seeds were greater than those of GA ones, and in the field, seeds obviously are exposed to alternat- IAA. In our study, there was no significant correlation between ing, not constant, temperatures (Baskin and Baskin 1998).
ABA content and seed germination. This is consistent with the Stratification at 5°C is optimal for seeds of many species that results of Ma et al. (2008a), which showed that high concentration require a moist low-temperature treatment for dormancy break of ABA did not inhibit germination of L.chinensis seeds. Thus, dor- (Stokes 1965). Zhang et al. (2006) showed that 10 weeks of mancy of L.chinensis seeds does not appear to be caused by ABA.
stratification on moist sand at 4°C improved germination ofL.chinensis seeds from 10 to 37.3% at 25/25°C. For control seeds Effects of maturity, treatment, temperature and of L.chinensis with different DAA in this study, highest germi- their interaction on seed germination of Leymus nation percentages were obtained mostly at 5/28°C. For naked- whole seeds with DAA > 20, high germination was obtained at Stage of seed development (seeds at different DAA), tempera- both 5/28°C and 5/35°C, and for naked-half seeds with DAA > tures, treatments on glumes and endosperm and their interac- 25, 100% germination was obtained at the three temperature tions showed significant effects on the germination percentage regimens (Figs 3–6). These results show that the dormancy of (GP) of L.chinensis seeds (Table 2) and the importance of the L.chinensis is controlled by glumes and endosperm and that dor- three factors on germination was as follows: treatment > ma- mancy could be broken by low/high (5/28°C) temperature to turity > temperature. During maturation, changes might take a greater extent than it could at 5/35°C. Perhaps, the seeds were place in seed structures, hardness of glumes, hormone content being cold stratified at the 5°C portion of the daily 5/28°C ther- and embryo activity that may affect seed germination to a great moperiod, whereas seeds at 5/35°C did not germinate well be- extent. Seed germination became sensitive to the removal and cause 35°C is too high for germination.
half-seed treatments mid-way through seed development, The seed coat is a multifunctional structure that plays an im- around 20–25 DAA. There were no significant differences in portant role in embryo nutrition during seed development and Journal of Plant Ecology later in protection of the embryo against detrimental agents from Baskin CC, Milberg P, Andersson L, et al. (2001) Seed dormancy-breaking the environment (Mohamed-Yasseen et al. 1994; Weber et al.
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chanical or chemical resistance. The seed coat restricts germi- Beaudoin N, Serizet C, Gosti F, et al. (2000) Interactions between absci- nation by being impermeable to water and/or oxygen or by its sic acid and ethylene signaling cascades. Plant Cell 12:1103–15.
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