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Published online 31 May 2007
Published in Crop Sci 47:1193-1205 (2007)
© 2007 Crop Science Society of America
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SEED PHYSIOLOGY, PRODUCTION & TECHNOLOGY

Influence of Soluble Sugars on Seed Quality in Nodulated Common Bean (Phaseolus vulgaris L.): The Case of Trehalose

Josué Altamirano-Hernándeza, Mercedes G. Lópeza, Jorge A. Acosta-Gallegosb, Rodolfo Farías-Rodríguezc and Juan José Peña-Cabrialesa,*

a Depto. de Biotecnología y Bioquímica, Cinvestav-U. Irapuato, Km. 9.6 Libramiento Norte, Carretera Irapuato-León, C.P. 36500, Gto. México
b Bean Program of INIFAP-Celaya, Km. 6, Carretera, Celaya-San Miguel Allende, Apdo. Postal 112, C.P. 38110 Celaya, Gto. México
c Instituto de Investigaciones Químico-Biológicas, UMSNH, Ciudad Universitaria, B-3, Morelia, Mich., México

* Corresponding author (jpena{at}ira.cinvestav.mx).


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Sucrose was the predominant sugar in seeds of five cultivars of common bean (Phaseolus vulgaris L.) grown under controlled conditions. The concentration of soluble sugars was higher in seeds from nodulated plants than seeds from N-fertilized or control (uninoculated and unfertilized) plants. Trehalose was detected in seeds but only from nodulated plants, and average values ranged from undetectable levels in seeds from control plants to 18 mg kg–1 in seeds from nodulated plants. Nodulated plants also produced larger and heavier seeds of higher quality; sucrose and trehalose contents were highly correlated with germination rate and with the vigor index of artificially aged seeds. Seeds of nine common bean cultivars grown in the field showed soluble sugar contents similar to those observed in seeds from nodulated plants raised in a growth chamber, except that the trehalose levels were significantly higher (23–111 mg kg–1). Trehalose was not detected in seeds from non-nodulating control plants. Seed trehalose level was significantly correlated with germination rate and with the vigor index of artificially aged seeds. Seed glucose concentrations were negatively related to all of the seed quality parameters used. In general terms, the recognition of the natural presence of trehalose in seeds has been limited to reports on the expression of the trehalose biosynthetic genes in a few plants. Our data show that in seeds produced by nodulated bean, plants trehalose occurs at high levels, suggesting that strategies to maximize nodulation may result in improvement of seed quality.


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TREHALOSE [{alpha}-D-glucopyranosyl-(1-1)-{alpha}-D-glucopyranoside] is a common disaccharide in microorganisms, where it acts as a compatible osmolite, providing protection against a variety of environmental stresses (Leslie et al., 1995; Desmarais et al., 1997; Eleutherio et al., 1997; Soto et al., 1999), as well as being plentiful in plant symbioses with fungi and bacteria (for review, see Mellor 1992). This, and the finding that trehalose is often toxic for plants (Veluthambi et al., 1981), led to the belief that sterile vascular plants do not contain trehalose. This view has been modified, however, as more sensitive detection techniques have been developed and on the discovery of trehalose biosynthetic genes in Arabidopsis (Leyman et al., 2001), although the overproduction of trehalose in transgenic seedlings (35S::AtTPS1) does cause a number of growth defects (Avonce et al., 2004). Thus, emphasis has shifted toward looking for a regulatory function for trehalose and its biosynthetic intermediates in plants, rather than for a protective function. In Arabidopsis for example, exogenous treatment with trehalose induced 48 transcripts and repressed 43 (Bae et al., 2005). More dramatically, and also in Arabidopsis, deletion of the tps1 gene (which encodes trehalose-6-phosphate synthase) resulted in the arrest of embryo development at the torpedo stage (Eastmond et al., 2002). Trehalose has also been implicated in the prolongation of viability of spores in Trichoderma (Pedreschi and Aguilera, 1997; Pedreschi et al., 1997); in the induction of desiccation tolerance and plant regeneration of barley (Hordeum vulgare L.) microspore-derived embryos (Ryan et al., 1999); in the tolerance of desiccation in pea (Pisum sativum L.) embryo protoplasts (Xiao and Koster, 2001); and in retarding senescence of gladiolus (Gladiolus spp.) petals (Yamada et al., 2003). Interestingly, an expressed sequence tag (EST), which was identified as tps1, was reported in the embryo proper and suspensor of Phaseolus coccineus L. (EST Project, 2005). Trehalose biosynthetic genes have not yet been reported in Phaseolus vulgaris L., however.

Trehalose is present in the root nodules of legumes (Streeter, 1985) and the concentration of trehalose in root nodules correlates well with whole-plant tolerance to drought (Farías-Rodríguez et al., 1998). In well-watered nodulated legumes, nodule trehalose concentrations are negatively correlated with N2 fixation (Streeter, 1985) and both nodule trehalose concentrations and nodule trehalase (EC 3.2.1.28, a trehalose hydrolyzing enzyme) activity fall toward nodule senescence (Müller et al., 1994b). Conversely, in nodules of drought-stressed plants, high concentrations of trehalose help to maintain efficient N2 fixation (Jiménez-Zacarías et al., 2004) and indeed the trehalose content of the nodules increases, with only a slight rise in nodule trehalase activity, as the plant develops (Streeter, 1985; Jiménez-Zacarías et al., 2004). It is not clear, however, what proportion of nodule trehalose is exported to plant tissues, affecting metabolism there, and what proportion is released on nodule senescence where it may, e.g., promote subsequent survival of the rhizobia in the soil (Müller et al., 2001; J.J. Peña-Cabriales, unpublished data, 2005).

Because of their intimate relationship with rhizobia that synthesize trehalose, legumes are important models to study the role of trehalose on plant metabolism. Based on the results of improved bean drought tolerance (Farías-Rodríguez et al., 1998; Jiménez-Zacarías et al., 2004), we hypothesized that trehalose accumulates in the aerial parts, including the seed, of nodulated legumes, where it may modify physiological parameters of agronomic importance.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plants and Rhizobia
Seeds of nine cultivars of the common bean were used: Canario 101, Flor de Mayo M38, Pinto Zapata, ICA Palmar, Negro Durango, BAT 477, DOR 364, BAT 477NN, and DOR 364NN (NN denotes non-nodulating). The strains of Rhizobium used (Fcin1, Fcin2, Fcin3, Fcin4, Fcin5, Fcin6, and Fcin7) were isolated from field-grown beans at Irapuato, Guanajuato, in central Mexico. Stock cultures were maintained at –70°C in PY medium (3 g L–1 yeast extract, 1 g L–1 CaCl2·2H2O, 5 g L–1 casein peptone), containing 50% (v/v) glycerol.

Growth Chamber Experiments
All seeds were surface sterilized with 2.5 M NaOCl for 5 min and rinsed with sterile, distilled water before use. To assess the effect of nodulation on trehalose content in the seed, an inoculated regime was established using a mixture of native rhizobial strains. The mixture was prepared by culturing independently each of the Fcin strains, whereupon equal amounts of log-phase cultures (~109 cells mL–1) were mixed and centrifuged at 8700 x g for 10 min, washed twice with a phosphate buffer (0.5 mM KH2PO4, 0.5 mM K2HPO4, pH 7), centrifuged as before, and resuspended in 50 mL of phosphate buffer. Fifteen seeds of cultivars Canario 101, Flor de Mayo M38, Pinto Zapata, ICA Palmar, and Negro Durango were inoculated by soaking the seeds for 1 h in this rhizobial suspension. Reinoculation was done at plant emergence by watering seedlings with 5 mL of similar rhizobial suspension prepared as before.

To assess the effect of plant N nutrition on soluble sugars in the seed, 15 seeds of each cultivar were used. Nitrogen fertilizer was provided to uninoculated plants watered with a Jensen solution [1 g L–1 Ca(H2PO4)2·H2O, 0.2 g L–1 K2HPO4, 0.2 g L–1 MgSO4·7H2O, 0.2 g L–1 NaCl, 0.1 g L–1 FeCl2·4H2O], and 1 mL of mineral solution (2.86 g L–1 H3BO3, 0.22 g L–1 ZnSO4·7H2O, 1,81 g L–1 MnCl2·7H2O, 0.09 g L–1 K2MoO4) containing 0.05% KNO3. Control treatments comprised uninoculated plants watered only with N-free Jensen solution.

Sand was sterilized in an oven for periods of 12 h at 250°C for three consecutive days, then used to fill 500-mL pots, which were then covered with brown paper and autoclaved three times, each for 1 h at 100 kPa, on three consecutive days. Plants were grown in a growth chamber with a 12-h day length at 22 ± 3°C. Seeds were harvested by hand at completion of the R9 physiological stage (seed maturity, approx. 67 d after planting).

Field Experiments
Field experiments (performed at the Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias, INIFAP-Celaya; lat. 20°31'40'', long. 100°48'55'', in a Pelic Vertisol soil) were conducted to compare seed sugar profiles with those obtained in the chamber experiment (Canario 101, Flor de Mayo M38, Pinto Zapata, ICA Palmar, and Negro Durango); reference cultivars BAT 477 and DOR 364 and their non-nodulating derivatives were also included. Plots (6 m2) were established for each of the nine cultivars and divided into three subplots (replicates). Field plants were fertilized with the recommended doses of N fertilizer for P. vulgaris (urea at 87 kg ha–1). Seeds were harvested by hand at the end of the R9 physiological stage (approx. 67 d after planting).

Soluble-Sugar Analysis
Three replicates of 10 seeds each from each cultivar and treatment were ground to a fine powder and 1 g of seed powder was used for analyses of soluble sugars. Total soluble sugars were extracted in boiling 80% (v/v) ethanol, centrifuged at 30800 x g for 15 min, and the supernatants set aside. Each pellet was reextracted twice, as before. The combined supernatants were evaporated to dryness before resuspension in 1 mL of 80% (v/v) ethanol. Aliquots (100 µL) of each sample were derivatized to obtain the aldonitrile peracetylated forms of the sugars (Macías-Rodríguez et al., 2002), which were analyzed by gas chromatography–mass spectometry (HP6850-HP5973) on an HP-5 MS column (Agilent Technologies, Palo Alto, CA). The operating conditions were: He carrier gas, 1 mL min–1; injector temperature, 300°C; injected volume, 1 µL with a 2:1 split; detector temperature, 300°C. The column was programmed to hold at 150°C for the first 3 min, then to increase in temperature at a rate of 6°C min–1 to a final temperature of 270°C, which was held for 15 min.

Identification of Trehalose
To confirm the identity of the putative trehalose peak, two independent assays were performed. The first method was by trehalose spiking (El-Bashiti et al., 2005): a trehalose standard was derivatized, and 4 ng were added to random samples; trehalose identification was confirmed when the putative trehalose peak increased after the trehalose standard was added. The second method was by addition of trehalase (EC 3.2.1.28, the trehalose hydrolyzing enzyme) to random samples: ethanolic extracts (100 µL) were dried and resuspended in 480 µL of malic buffer (50 mM malic acid at pH 6, 1 mM benzamidine·HCl, 0.5 mM PMSF, 0.44 M ß-mercaptoethanol), 50 microunits of trehalase (one unit of trehalase activity is the amount of enzyme that converts 1 µmol of trehalose to 2 µmol of glucose per minute at pH 5.7 at 37°C; Sigma-Aldrich, St. Louis, MO) was added and incubated for 45 min at 37°C, after which the reaction was stopped by placing the tubes in boiling water for 10 min; the samples were then dried and derivatized as described above. The presence of trehalose was confirmed when the putative trehalose peak disappeared and the glucose peak increased by a corresponding amount.

Germination
Thirty seeds of each cultivar per treatment were incubated in wet paper rolls in the dark at 25°C. On the fifth day, germination was assessed.

Seedling Vigor Index
Following the method of Varghese and Naithani (2000), root lengths of all germinated seeds were measured at 3 d from the day of germination and the seedling vigor index (SVI) derived by applying the following formula: SVI = germination (%) x average root length (cm).

Artificial Seed Aging
Seed quality was evaluated by an artificial aging assay (modified from Machado Neto et al., 2001). Three lots of 10 seeds were placed in lines inside open plastic containers and incubated in a dryer with 300 mL of distilled water at 45°C for 72 h and 100% relative humidity, then dried to a constant weight at 35°C. Germination percentage and seedling vigor index were then measured as described above.

Statistical Analysis
Seed-weight and seed-size data were analyzed as a two-factorial analysis (Factor A represents the number of seed cultivars used, Factor B represents plant treatments). Germination and vigor index were analyzed as a three-factorial analysis (Factor A represents the number of seed cultivars used; Factor B represents plant treatments; Factor C represents seed aging). All other data were analyzed as a completely random design with three replicates. Factorial ANOVA was performed with the STATISTICA data analysis software system (Version 7.0, StatSoft Inc., Tulsa, OK). Correlation coefficients were calculated with the STATISTICA system at different significance levels (P = 0.1, 0.05, and 0.01) to assess the degree of interaction between the different evaluated parameters.


   RESULTS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Confirmation of the Presence of Trehalose
Trehalose was identified by mass spectroscopy (data not shown), and its presence in bean seeds was confirmed by spiking assays. The gas chromatography analysis trace of crude extracts of ICA Palmar seeds is shown in Fig. 1a and the putative trehalose peak, with a retention time of 30.5 min, is next to the sucrose peak. To unambiguously assign this peak to trehalose, Fig. 1b shows the effect of spiking with commercial trehalose, resulting in an increase in the size of the peak at 30.3 min. Furthermore, after trehalase was added to crude extracts (Fig. 2), the putative trehalose peak decreased dramatically in area with a concomitant increase in the glucose peak (13 min).


Figure 1
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  		Figure 1. Gas chromatography (GC) chromatograms of trehalose spiking assays: (a) chromatogram of a crude ethanolic extract of cultivar ICA Palmar seeds and (b) chromatogram of a similar cultivar ICA Palmar ethanolic extract containing trehalose. Insets: enlargements of areas of trehalose peaks; arrows indicate trehalose peak. *Unknown disaccharide.

 

Figure 2
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  		Figure 2. Gas chromatography (GC) chromatograms of trehalase hydrolysis assays: (a) chromatogram of a crude ethanolic extract of cultivar ICA Palmar seeds and (b) chromatogram of a crude ethanolic extract of cultivar ICA Palmar seeds after treatment with the enzyme trehalase. Insets: enlargements of areas of trehalose peaks; arrows indicate trehalose peak. *Unknown disaccharide.

 
Growth Chamber Experiment
Seed Morphology
Experiments from growth chambers (summarized in Table 1) showed that seeds from control bean plants (uninoculated and unfertilized) were significantly lighter than those obtained from KNO3–fertilized or nodulated plants, and those seeds from nodulated plants, especially Pinto Zapata and ICA Palmar cultivars, were heavier than from N-fertilized plants. The average seed weight values were 191.2 ± 7 mg seed–1 in control plants, 312.4 ± 41.3 mg seed–1 in fertilized plants, and 368.4 ± 40.3 mg seed–1 in nodulated plants. Negro Durango produced the heaviest seeds amongst the cultivars tested, being overshadowed only by seeds from nodulated Canario 101. Seeds from control plants were always significantly smaller than those from fertilized or nodulated plants and seeds from nodulated plants were always larger than seeds produced by N-fed plants, with the exception of Flor de Mayo M38, which produced seeds of statistically similar size under all treatment regimes; the average seed size was 0.94 ± 0.03 cm seed–1 in control plants, 1.14 ± 0.14 cm seed–1 in fertilized plants, and 1.18 ± 0.15 cm seed–1 in nodulated plants. Negro Durango produced the largest seeds in control and nodulated plants, being matched only by seeds from Pinto Zapata (in fertilized plants only).


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  		Table 1. Average dry weight and size of seeds produced by common bean (Phaseolus vulgaris L.) plants (N fertilized vs. nodulated) grown in a growth chamber.

 
Seed Germination
The germination capability of seeds is summarized in Table 2; under standard germination conditions, seeds from control plants (unfertilized and uninoculated) showed the lowest germination percentage compared with seeds from KNO3–fertilized or nodulated plants. Seeds from nodulated plants showed the highest germination percentage. Maximum germination values were of 60% in seeds from control plants, 90% from fertilized plants, and 97% from nodulated plants.


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  		Table 2. Effect of artificial aging on common bean (Phaseolus vulgaris L.) seed quality.

 
Seed quality was evaluated by an artificial aging assay, resulting in the reduction of germination in all the cultivars tested (Table 2). Seeds from control plants were more sensitive to this process (12% germination) than seeds from fertilized or nodulated plants (71 and 80% germination, respectively).

Vigor Index of Seedlings
Seedling vigor was computed as a function of germination percentage and the growth rate of roots (Table 2). Larger numbers indicated faster and more vigorous development of seedlings. Seeds from control plants showed the lowest seedling vigor index, with an average value of 38 ± 12. Seeds from fertilized plants had an average vigor index of 489 ± 27. Seeds from nodulated plants showed the highest index, with an average value of 554 ± 55. Seeds from control plants were the most affected by artificial aging, with an average vigor index of 7 ± 1. Artificially aged seeds from fertilized plants showed an average index of 337 ± 31. Seeds produced by nodulated plants were most resistant to artificial aging, maintaining an average index of 432 ± 19.

Sugar Content in Seeds from Plants Raised in Chambers
The principal sugar in all seeds obtained from the growth chamber experiment was sucrose, (100-fold higher than any other), followed by glucose (see Table 3). With the exception of fructose, the average content of soluble sugars was higher in seeds from fertilized plants than control plants. The sugar contents in seeds from nodulated plants, however, increased compared with seeds from fertilized plants, the major increase being in the content of trehalose, myo-inositol, and sucrose.


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  		Table 3. Effect of nodulation and N fertilizer on soluble sugar contents of seeds obtained from different cultivars of common bean (Phaseolus vulgaris L.) grown in a growth chamber.

 
Seeds from control plants contained 418 mg kg–1 glucose, 119 mg kg–1 myo-inositol, 56 mg kg–1 fructose, 20 g kg–1 sucrose, and trehalose was under detectable levels. Seeds from fertilized plants contained 433 mg kg–1 glucose, 165 mg kg–1 myo-inositol, 52 mg kg–1 fructose, 24 g kg–1 sucrose, and 1 mg kg–1 trehalose. Seeds from nodulated plants contained 516 mg kg–1 glucose, 294 mg kg–1 myo-inositol, 55 mg kg–1 fructose, 39 g kg–1 sucrose, and 18 mg kg–1 trehalose.

Correlation analysis among the different soluble sugars in seeds from control plants showed no statistically significant coefficients among them (Table 4). In seeds from fertilized plants (Table 5), correlation coefficients were significant among sucrose and fructose (r = 0.88, P < 0.05), glucose and myo-inositol (r = –0.82, P < 0.1), and trehalose and fructose (r = –0.81, P < 0.1). Correlation coefficients in seeds from nodulated plants (Table 6) were of low significance between fructose and myo-inositol (r = 0.86, P < 0.1) and highly significant among sucrose and trehalose (r = 0.99, P < 0.01).


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  		Table 4. Correlation values among the different soluble sugars in the common bean (Phaseolus vulgaris L.) seeds obtained from control plants (uninoculated and without N fertilization; n = 5).

 

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  		Table 5. Correlation values among the different soluble sugars in common bean (Phaseolus vulgaris L.) seeds obtained from N-fertilized plants (n = 5).

 

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  		Table 6. Correlation values among the different soluble sugars in common bean (Phaseolus vulgaris L.) seeds obtained from nodulated plants (n = 5).

 
Correlation graphics among trehalose with myo-inositol and sucrose of seeds from fertilized and nodulated plants are shown in Fig. 3; trehalose correlation with myo-inositol shifted from a significant positive slope in the seeds from fertilized plants (Fig. 3a) to a nonsignificant negative slope in seeds from nodulated plants (Fig. 3b). Also, the correlation slope between trehalose and sucrose shifted from a negative nonsignificant slope in seeds from fertilized plants to a highly significant positive correlation slope in seeds from nodulated plants.


Figure 3
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  		Figure 3. Correlation graphics between trehalose and soluble sugars from common bean (Phaseolis vulgaris L.) seeds obtained from (A) fertilized plants and (B) nodulated plants raised in a growth chamber. Solid lines are the slope of the correlation coefficient; dotted lines are the confidence limits.

 
Table 7 shows a general analysis from the growth-chamber experiment where significant correlation coefficients for trehalose with myo-inositol and sucrose (r = 0.49, P < 0.1; and r = 0.79, P < 0.01, respectively), and between sucrose and myo-inositol (r = 0.5, P < 0.1) were obtained.


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  		Table 7. Correlation values among the different soluble sugars in bean (Phaseolus vulgaris L.) seeds obtained under growth chamber conditions (n = 15).

 
Correlation Analysis
Table 8 shows the correlation coefficients obtained among soluble sugar concentrations and seed parameters (seed weight and size, germination, and vigor index from normal and artificially aged seeds). In general terms, all glucose coefficients in seeds from control and N-fed plants were negative, while in seeds from nodulated plants, coefficients were only negative with regard to seed morphological parameters (seed weight and size). The opposite trends were observed in myo-inositol coefficients, which were positive in seeds from control or N-fed plants, and were negative in seeds produced by nodulated bean plants. Statistically significant correlation coefficients in control plants' seeds were observed among glucose with seed weight (r = –0.81, P < 0.1) and vigor index (r = -0.93, P < 0.05), in fertilized plants' seeds among glucose with seed weight and germination of aged seeds (r = –0.92, P < 0.05; r = –0.88, P < 0.1, respectively), and in nodulated plants' seeds among sucrose and trehalose with vigor index (r = 0.85 and 0.79, P < 0.1).


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  		Table 8. Correlation coefficients among soluble sugars and seed parameters of different common bean (Phaseolus vulgaris L.) cultivars (n = 15).

 
Table 9 shows the correlation analysis among seed parameters. In seeds of control plants, most of the correlations concerning the germination of aged seeds were negative, while in seeds from fertilized and nodulated plants all correlations were positive. Seed weight correlated significantly with seed size of seeds from control and nodulated plants (r = 0.88, P < 0.05, in both cases). Also, seed weight from control plants correlated significantly with their vigor index (r = 0.83, P < 0.1), while in seeds from fertilized plants, correlations with germination percentage of artificially aged seeds were seen (r = 0.91, P < 0.05). In seeds from nodulated plants, seed quality parameters (germination, vigor index, and germination of aged seeds) correlated highly with the vigor index of aged seeds (P < 0.05).


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  		Table 9. Correlation coefficients between bean (Phaseolus vulgaris L.) seed morphological parameters (n = 15).

 
Field Experiments
Large populations of native rhizobia are common in Mexican soils, making it difficult to obtain bean plants that are free of nodules. Thus to obtain field-grown nodule-free plants, the non-nodulating cultivars BAT 477NN and DOR 364NN were included in trials along with their nodulating progenitors (BAT 477 and DOR 364), as reference cultivars.

Seed Morphology
Among the five cultivars common in both experiments, the average weight per seed obtained from nodulated field plants was 395.2 ± 0.15 mg seed–1, a higher value than seeds produced by nodulated plants obtained in the growth chambers. Interestingly, seed weights of reference nodulating cultivars (BAT 477 and DOR 364) were higher than their non-nodulating counterparts (BAT 477NN and DOR 364NN), although this was clearly significant only in the case of BAT 477 (Table 10).


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  		Table 10. Morphological and physiological parameters of common bean (Phaseolus vulgaris L.) seeds obtained from plants raised under field conditions.

 
Similarly, seed size was significantly higher in nodulated field plants (average seed size of 1.35 ± 0.15 cm seed–1) than their nodulated counterparts grown in the growth chamber. Seeds from BAT 477 were significantly larger than those from its non-nodulating counterpart, whereas the DOR 364 cultivars showed no difference (Table 10).

Seed Germination
Standard germination of cultivars grown in growth chambers and field experiments were very similar (95%). Germination values of BAT 477 and DOR 364 were higher than their non-nodulating counterparts, but still fell within a broadly similar range to that observed in the comparison between seeds from fertilized and nodulated plants from the growth chamber experiment (Table 10).

Seeds from plants raised in the field were least affected by the artificial aging, with an average decrease in germination of only 12%. Seeds from nodulating BAT 477 and DOR 364 still maintained a higher germination rate than those observed from their non-nodulating counterparts (Table 10).

Vigor Index of Seedlings
Seeds from nodulated field plants showed a higher vigor index than their nodulated counterparts from the growth-chamber experiment, with an average value of 621 ± 49. Reference cultivars BAT 477 and DOR 364 showed higher index values than their non-nodulating counterparts (Table 10). After artificial aging, seeds from nodulated field plants showed an average vigor index of 402 ± 47, a lower value than their counterparts in the growth chamber. The vigor index of seeds from nodulating reference cultivars (BAT 477 and DOR 364) were less affected by the artificial aging than their non-nodulating derivatives (Table 10).

Sugar Content of Seeds from Field-Grown Plants
The principal sugar in seeds obtained from field plants was sucrose (100-fold higher than any other), followed by glucose and myo-inositol (Table 11). Seeds from field plants contained 548 mg kg–1 glucose, 450 mg kg–1 myo-inositol, 65 mg kg–1 fructose, 29 g kg–1 sucrose, and 53 mg kg–1 trehalose. The myo-inositol and trehalose contents of seeds from nodulated field plants were higher than their nodulating counterparts grown in the growth chamber. Reference cultivars BAT 477 and DOR 364 showed a significantly lower glucose content in nodulating varieties than in non-nodulating ones. On the other hand, the myo-inositol content of seeds of nodulating varieties was significant higher than in non-nodulating varieties. Trehalose was only observed in seeds from nodulating varieties.


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  		Table 11. Sugar contents in seeds obtained from different cultivars of common bean (Phaseolus vulgaris L.) grown under field conditions.

 
Correlation coefficients for sugars in seeds obtained under field conditions were significant only between sucrose and trehalose (r = 0.61, P < 0.1; Table 12).


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  		Table 12. Correlation values among the different soluble sugars in the bean (Phaseolus vulgaris L.) seeds obtained under field conditions (n = 9).

 
Correlation Analysis
Table 13 provides correlation analysis among soluble sugars and seed parameters from the field experiment, where more significant correlation coefficients were obtained than with experiments using growth chamber plants. Glucose and fructose were negatively related to most seed parameters tested (from r = –0.52 to r = –0.85, P < 0.01), whereas myo-inositol coefficients were mostly positive, resembling those observed in seeds from fertilized growth chamber plants rather than from nodulated plants. Sucrose correlation coefficients were significant only with seed size and vigor index (r = 0.62 and 0.65, respectively, P < 0.1). Trehalose coefficients were significantly correlated with the vigor index of normal seeds (r = 0.61, P < 0.05) and with the germination rate and vigor index of aged seeds (r = 0.61, P < 0.1; r = 0.73, P < 0.05, respectively).


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  		Table 13. Correlation coefficients among soluble sugars and seed parameters of seeds from common bean (Phaseolus vulgaris L.) field plants (n = 15).

 
Table 14 shows the correlation analysis within seed parameters from the field experiment. Interestingly, all parameters were significantly correlated (values ranging from r = 0.83 to 0.94, P < 0.01), with the exception of seed weight with vigor index of artificially aged seeds. On average, seed size was more highly correlated with other quality parameters (germination and vigor index from normal and artificially aged seeds) than was seed weight.


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  		Table 14. Correlation coefficients among seed morphological parameters from common bean (Phaseolus vulgaris L.) field plants (n = 15).

 

   DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Pacovsky and Fuller (1988) reported that soybean [Glycine max (L.) Merr.] seeds produced by plants infected with Bradyrhizobium and Glomus were of higher quality than those obtained from well-fertilized plants and that this effect was not associated with the enhanced plant nutritional status derived from these symbioses, as both types of seed had similar contents of N and P. These results highlighted benefits accruing from the mycorrhiza–rhizobia–legume symbiosis, which are independent of the enhanced nutritional status of the plant and obtained by biological N2 fixation and P uptake. Thus reports of the accumulation of high levels of trehalose in legume nodules (Streeter, 1980; Kouchi and Yoneyama, 1986) were greeted with great interest because the survival to desiccation by numerous species had been attributed to trehalose. Since then, the synthesis of trehalose by rhizobia has been related with various other characteristics including nodulation competence (Jensen et al., 2005), the tolerance of common bean to drought (Farías-Rodríguez et al., 1998), and nodule functionality under drought conditions (Jiménez-Zacarías et al., 2004).

Before seed germination, maturation drying is normally required, during which important metabolic changes occur, including the accumulation of compounds thought to increase the capacity of seeds to resist desiccation. Trehalose, however, has previously been thought to be absent from seeds (Derek and Black, 1994) and that it is other sugars, especially raffinose and sucrose, that accumulate in zones where they confer high viscosity and contribute to the structural integrity of dry cells. This combination helps to slow down enzymatic reactions and to protect seeds from further desiccation (Derek and Black, 1994; Bernal-Lugo and Leopold, 1998). Interestingly, Arabidopsis thaliana (L.) Heynh. carries genes implicated in trehalose synthesis (Blázquez et al., 1998; Vogel et al., 1998), and deletion of the tps1 gene (encoding trehalose-6-phosphate synthase) is lethal to embryo development (Eastmond et al., 2002). On the other hand, the use of a dexamethasone-inducible transcription system, which permits the recovery of tps1 mutant plants, showed that induction was necessary for flowering to occur (van Dijken et al., 2004). Furthermore the embryo and suspensor of Phaseolus coccineus L., the scarlet runner bean, contain an expressed sequence tag (EST) of a putative TPS1 gene (EST Project, 2005).

We report for the first time that trehalose accumulates naturally in seeds from nodulated P. vulgaris. Our primary working theory is that this trehalose originates from root-nodule bacteroids, since seeds from control, non-nodulated or non-nodulating plants lacked the sugar, or contained only traces, which may have been due to stray mychorrhizal infections, especially in the field-grown material. Transport mechanisms (both across membranes, as well as systemically in the plant) for trehalose have still not been elucidated, but we assume that trehalose produced by the rhizobial bacteroids is partitioned between the nodule and the seed during pod filling, which, in many legumes, is the stage where the nodules begin to senesce and trehalose becomes the most abundant carbohydrate there (Müller et al., 2001). Further support can be found in the report of Jiménez-Zacarías et al. (2004), which described the tendency for nodules to accumulate more trehalose at the flowering stage (i.e., the stage in the growth cycle immediately before pod filling), even under conditions of drought. In agreement with this is the results of a study by Altamirano-Hernández et al. (2004), who found that the nodules of legumes in a tropical deciduous forest ecosystems accumulated trehalose at very high rates at the beginning of the dry season, which again correlates with flowering. Trehalose concentrations in seeds from nodulated plants are quite variable, but such variation has also been reported in nodules (Farías-Rodríguez et al., 1998; Jiménez-Zacarías et al., 2004) and thus, assuming that the trehalose is produced symbiotically, such variation could well be due to other features of the legume–rhizobia association. Nevertheless, Bae et al. (2005) treated Arabidopsis with exogenous trehalose and found an increase in trehalose content in the seedlings. Although they proposed a possible transport from the medium, through the roots, to the leaves, it is still possible—arguing against our primary working theory—that exogenous trehalose can stimulate the plants to biosynthesize its own trehalose to detectable levels. Thus, in our study, trehalose from bacteroids may have induced the bean plants to biosynthesize endogenous trehalose in their seeds. Although the presence of trehalose normally stimulates trehalase (i.e., trehalose hydrolysis [see, e.g., Veluthambi et al., 1981]) and not a positive feedback loop involving further trehalose synthesis, it is interesting that, in parallel work, we have identified putative TPS1 (i.e., synthetic) genes in P. vulgaris; the sequences are presently under analysis to confirm their identity and functionality (J.J. Peña-Cabriales, unpublished work, 2006). It has been reported that the activity of trehalase is regulated by phytohormones: auxins increase trehalase activity, whereas anti-auxins have a negative effect (Müller et al., 1995). High concentrations of abscisic acid (ABA) are found in developing legume seeds (Derek and Black, 1994), and it may very well be that increased ABA levels can depress trehalase activity at around pod-fill time.

Exposure to 20 mM NO3 caused a decrease in the levels of trehalose and starch in nodules while the level of sucrose increased (Müller et al., 1994a). Also, the addition of validamycin A (an inhibitor of trehalase) caused an increase in nodule trehalose content and a concomitant decrease in the sucrose and starch pools (Müller et al., 1995), implying a negative correlation between trehalose and sucrose in the nodule, perhaps by drawing the common precursor, glucose, from the same pool. In seeds from fertilized plants, we observed that sucrose was negatively correlated with trehalose (r = –0.74) but the opposite was observed in seeds from nodulated plants: sucrose and trehalose were significantly correlated (r = 0.99), in agreement with a report on Arabidopsis in which added trehalose (i.e., exogenous trehalose treatment) promoted an increase in sucrose content as well as in starch (Bae et al., 2005). Interestingly, under field conditions, trehalose was not detected in the seeds of the Negro Durango cultivar, which resembles the behavior observed in fertilized plants in the growth chamber. This cultivar was developed in the northern semiarid region of Mexico, a region characterized by shallow soils low in N, P, and organic matter. Therefore, the observed response might be due to the fact that this cultivar is highly efficient in nutrient uptake, and the addition of N fertilizer in the field may have reduced trehalose contents in the nodules as reported (Müller et al., 1994a). Under field conditions, seeds from nodulated cultivars have more trehalose than in the growth chamber; this higher trehalose content may be the result of transitory stresses that may occur under field conditions (mainly drought or osmotic), which promotes the increase of trehalose in the nodules (Farías-Rodríguez et al., 1998; Jiménez-Zacarías et al., 2004). Nevertheless, further work is needed to clearly elucidate how N fertilization in combination with stress under field conditions affects trehalose metabolism in this symbiosis.

Seeds from nodulated plants also contained higher concentrations of myo-inositol, a polyol that apparently can play a multitude of roles; it has been implicated in the synthesis of phytic acid in seeds (Hegeman et al., 2001; Shi et al., 2005), but is also a precursor of compounds that participate in signal transduction, stress protection, hormonal homeostasis, and cell-wall synthesis (Hegeman et al., 2001). Furthermore, myo-inositol can be readily catabolized by rhizobia, and the capability to use inositol derivatives has been correlated with nodulation and N2 fixation (Jiang et al., 2001). myo-Inositol also affects the synthesis of raffinose, a trisaccharide involved in the tolerance of seeds to desiccation (Bernal-Lugo and Leopold, 1998) and indeed the suppression of 1-D-myo-inositol-3-phosphate synthase by an antisense RNA construct in transgenic potato (Solanum tuberosum L.) reduced raffinose levels in leaves by 12% (Keller et al., 1998). Experiments in which myo-inositol was fed to peas failed, however, to clarify its role (Karner et al., 2004) and thus the significance of myo-inositol levels in bean seeds still needs to be elucidated.

We have also shown that seeds produced by nodulated plants in growth chamber experiments had higher germination rates and vigor indices than N-fed plants. High variability was observed in seedling development from plants raised in the field, which translated to a lower vigor index of aged seeds, in comparison to seeds from nodulated plants raised in the growth chamber. Nevertheless, this effect could be the result of challenging conditions normally found in the field (insect predation, pathogen attack, moisture deficiency, etc.) that may negatively affect seed quality. Interestingly, seeds from field-grown BAT 477 and DOR 364 showed better seed quality than did their non-nodulating derivatives, supporting direct support for the theory that the root-nodule symbiosis confers additional benefits over and above those resulting from biological N2 fixation (Pacovsky and Fuller, 1988).

In agreement with previous reports (Wettlaufer and Leopold, 1991; Bailly et al., 2001), we observed that the accumulation of monosaccharides, in particular glucose, was negatively correlated with common seed-quality parameters and it has been postulated that, as a reducing sugar, glucose may participate in Maillard reactions (Wettlaufer and Leopold. 1991).

Consistent with the findings of Siddique and Goodwin (1980) and of Bretagnolle et al. (1995), we found that the bean cultivars Negro Durango and Canario 101 produced large seeds with high quality, but that seeds from nodulated Canario 101 were of a slighter better quality than those from nodulated Negro Durango under controlled-growth conditions, whereas the opposite was observed in seeds produced by these two cultivars under field conditions. This cannot be explained by trehalose content alone, since the trehalose content of Negro Durango seeds was lower than that of Canario 101 under both conditions. Higher quality of Negro Durango seeds could be due to a more efficient nutrient uptake, as mentioned above, which can be translated to a high accumulation of reserve compounds in the seed.

The cultivar ICA Palmar, which produces smaller, lighter seeds, under field conditions, did have the highest trehalose content, however, and showed the best seed quality. These observations may suggest two different physiological (and certainly genotypic) strategies to produce seeds of high quality. Interestingly, it has been argued that for optimal quality, seeds must develop and mature at cool temperatures in a dry environment (Siddique and Goodwin, 1980), and in Mexico, where bean production is mainly under rain-fed conditions, these conditions prevail. As we have previously documented, nodulated common bean plants are more tolerant of water deprivation than are plants without nodules (Farías-Rodríguez et al., 1998; Jiménez-Zacarías et al., 2004) and this desiccation protection could well contribute, in some cultivars, to increased seed longevity.

It is well known that high levels of oligosaccharides from the raffinose family (RFOs) are accumulated in seeds and are associated with seed quality and storability (Steadman et al., 1996; Bernal-Lugo and Leopold, 1998; Bailly et al., 2001). Unfortunately, however, the presence of RFOs in seeds has a negative effect on digestibility, because humans lack {alpha}-galactosidase. To address this problem, the genetic manipulation of bean has been suggested to reduce the RFOs by post-harvest expression of the {alpha}-galactosidase gene from Thermotoga neapolitana (Wang et al., 2003). It will be of great interest to determine if, in nodulated bean plants, an increase in myo-inositol, which plays important roles in the synthesis of RFOs (Peterbauer and Richter, 2001) and trehalose levels in seeds, could mean a decrease in RFOs without affecting seed quality.

Our results show that nodulation is a stringent prerequisite for trehalose content and a lax requisite for high myo-inositol content in bean seeds. Developments in artificial seed technology have shown that the addition of trehalose has a positive effect on seed survival (Ryan et al., 1999) and here we have demonstrated that nodulated bean plants produced seeds with higher trehalose content than did N-fed plants. These seeds were of better quality than those produced from non-nodulated counterparts and this, in the field, may translate into higher yields. The amount of trehalose in seeds is related to the bean genotype and to prevailing nodulation conditions, and perhaps also, as observed in nodules, to soil moisture conditions. Exactly how these changes occur and the roles they play in seed development and longevity remain to be elucidated and thus our future work will focus on the identification of seed metabolic pathways that are modified by trehalose in nodulated plants.


   ACKNOWLEDGMENTS
 
This work was supported by CONACYT.


   NOTES
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 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Received for publication September 8, 2006.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 





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