Control of storage protein accumulation during legume seed development

J. Plant Physiol. 158. 457 – 464 (2001)  Urban & Fischer Verlag http://www.urbanfischer.de/journals/jpp

Control of storage protein accumulation during legume seed development* Sabine Golombek1, Hardy Rolletschek, Ulrich Wobus, Hans Weber** Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), D-06466 Gatersleben, Germany 1

Present address: Universität Kassel, Institut für Nutzpflanzenkunde, D-37213 Witzenhausen, Germany

Received September 30, 2000 · Accepted January 12, 2001

Summary The regulation of partitioning of carbohydrate skeletons into different storage products of developing seeds is still not understood. We explored two ways to gain more insight in the process. First we analyzed mechanisms that control storage protein accumulation in Vicia faba seeds of contrasting protein content. As expected, the seeds of the high protein genotypes (HP) contained more protein and total nitrogen as compared to the low protein genotypes (LP) whereas starch and total amounts of carbon were not altered. There was no major difference in the proportion of amino acids delivered from seed coats into the endospermal cavity of either HP or LP genotypes. However, HP genotype cotyledons contained two-fold higher levels of free amino acids at the later developmental stages when their higher protein content was expressed. After four hours of incubation, in vitro uptake rates of 14C glutamine by HP cotyledons were significantly higher for the protein rich cotyledons indicating a possible higher capacity to take up amino acids. In both genotypes 14C-glutamine was rapidly converted into glutamate and then partly decarboxylated to γ-amino butyric acid. However, in the HP cotyledons the ratio of 14C-glutamine to 14C-glutamate was higher as compared to the LP cotyledons reflecting the observed higher glutamine uptake rate. In a second approach we studied Vicia narbonensis seeds which expressed ADP glucose pyrophosphorylase in antisense orientation. These seeds contained less starch and more sucrose and water but also more protein. In addition, blocking the starch synthesis pathway caused pleiotropic effects on water content and induced temporal changes in seed development. The resulting longer seed fill duration period could partially explain the observed elevated protein content in the AGP-antisense seeds.

Key words: assimilate partitioning – storage protein – amino acid metabolism – nutrient transport – legume seed development Abbreviations: AAP amino acid permeases. – AGP ADP-glucose pyrophosphorylase. – GABA γ-aminobutyric acid. – HP high protein. – LP low protein. – PEPC phosphoenolpyruvate carboxylase * This paper is more focussed than the oral presentation at the meeting since a more general review has been published recently (Wobus and Weber 1999). ** E-mail corresponding author: [email protected] 0176-1617/01/158/04-457 $ 15.00/0

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Introduction Legume seeds synthesize both proteins and starch and represent the most important source of protein for human and animal nutrition. For example Vicia faba seed protein is in the range of 25 to 33 % of dry weight (Müntz et al. 1986). It is therefore of great interest to evaluate mechanisms that control storage protein accumulation and content in legume seeds. Seed proteins are predominantly synthesized in the cotyledons during the mid to late stages of development. Amino acids as precursors are unloaded from the phloem into the maternal seed tissues from where they are transferred to the seed apoplast and subsequently taken up by the symplasmically isolated embryo. Storage protein synthesis is regulated at different levels. The most important ones are availability and partitioning of assimilates and nitrogen compounds and the genetic properties of the cultivar. During seed growth, nutrients like sugars and nitrogen compounds confer regulatory control on storage activities (for reviews see: Weber et al. 1997, 1998 a, Wobus and Weber 1999). Composition of seed storage products can be regulated by the supply of nutrients. The endosperm-specific synthesis of storage proteins in maize (Balconi et al. 1991) and barley (Müller and Knudsen 1993) was shown to be under nutritional control and dependent on nitrogen availability. Legume embryos can be grown in vitro using relatively simple media including sucrose, one amino acid and minerals. The synthesis of storage products therefore, seems to be exclusively regulated by the embryo itself rather than by the maternal plant. Soybean seeds need only a minimal supply of nitrogen to maintain normal dry weight accumulation indicating that genetic differences in the percentage of accumulated protein seem to be regulated by the cotyledons and not by the external supply of nitrogen (Hayati et al. 1996). Several lines of evidence suggest that for storage protein synthesis it is essential to make amino acids or reduced nitrogen available to the embryo or endosperm (Motto et al. 1997). Barratt (1982) found significant correlations between seed protein and free amino acid concentration in the cotyledons, therefore, the presence of specific mechanisms for uptake and partitioning of nitrogen compounds could be important control factors. Amino acid uptake into embryos of soybean and pea was analyzed by Bennett and Spanswick (1983) and DeJong et al. (1997). These authors found a non-saturable, diffusion-like mechanism of uptake and, in addition, a saturable component, which represented most probably a H + -amino acid co-transport. The latter was inducible by low nitrogen and its relative contribution to the total uptake increased during seed development (Bennett and Spanswick 1983). Particular isoforms of amino acid permeases (AAP family) in Arabidodpsis were specifically expressed in the seeds (Hirner et al. 1998). Amino acid uptake into parenchyma cells of pea cotyledons could probably be mediated by specific H + -amino acid transporters (Tegeder et al. 2000, Miranda et al. in preparation). How-

ever, a rate-limiting role of such seed-specific transporters have not been proven yet. Glutamine and/or asparagine are preferentially imported into legume cotyledons (Miflin and Lea 1977). The biosynthesis of other amino acids in the seeds involves glycolysis and tricarbonic acid cycle products to provide carbonskeletons. Some carbon derived from sucrose must therefore be diverted to the synthesis of storage proteins (Turpin and Weger 1990). This raises the question as to whether and how intimately the pathways of starch and protein biosynthesis may be connected and what the control points could be. There is evidence that carbon partitioning at least in leaves is controlled in response to nitrogen availability. Thereby, the flow of carbon into starch increases or decreases in response to low or high nitrogen through an effect on the anaplerotic reaction via phosphoenolpyruvate carboxylase (PEPC) (Champigny et al. 1992). A recent characterisation of PEPC in developing seeds of V. faba indicated a similar key regulatory role in the anaplerotic carbon flow during storage protein synthesis (Golombek et al. 1999). It is of interest to know whether increasing or decreasing one storage component leads to a compensatory change of the other form. There is physiological evidence that C and N metabolism in developing soybean seeds are not tightly linked (Hayati et al. 1996) and that the rate and duration of protein deposition in wheat endosperm seems to be independent of starch biosynthesis (Jenner et al. 1991) but at least the final grain N concentration is determined by the N/C ratio transported to the ear. Therefore, it is thought that both pathways are not directly connected (Barneix et al. 1992). However, in pea seed mutants affected in starch formation also storage protein accumulation is affected suggesting a linked biosynthesis of these products (Casey et al. 1998). Wrinkled pea seed mutants with reduced starch have increased protein contents on a per gram basis (Perez et al. 1993, Boutin et al. 1998) indicating a change in partitioning between starch and protein. In order to study mechanisms and strategies of genotypes which accumulate high or low amounts of seed proteins we made use of the large collection of Vicia faba accessions in the Genebank, IPK Gatersleben, and selected genotypes which differ in their seed protein content. We concentrated on mechanisms in the seed and measured amino acids in seed tissues and analyzed the partitioning after feeding 14C glutamine. In addition we described transgenic V. narbonensis seeds in which starch production has been downregulated. We provide evidence for a possible role of uptake mechanisms to control seed protein accumulation. However, changes in developmental processes like the duration of the seed filling period could also contribute to the higher protein content.

Control of seed storage protein accumulation

Materials and Methods

Results

Plant material

Description of genotypes differing in seed protein content

Seeds were obtained from the Genebank, Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), Gatersleben, Germany. Plants were grown in growth chambers under a regime of 16 h light and 8 h dark at 20 ˚C. After harvest seeds were immediately chilled on ice.

Extraction and determination of free amino acids, sugars, starch and total C and N Plant samples were homogenized and extracted with 80 % ethanol. Samples including standards were derivatized using the AccQ-Tag method (Waters, USA) and run on a HPLC system (GP40 Gradient module, AD20 Absorbance detector, Dionex, USA) using a NovaPak-C18 column (3.9 × 150 mm; Waters). Separation was carried out at 37 ˚C with a gradient of acetate/phosphate buffer and acetonitrile/ water. Sugars and starch were measured enzymatically as described in Heim et al. (1993). To determine total nitrogen and carbon plant material was dried at 80 ˚C to a constant weight and pulverised using a ball-mill. The tissue concentration of total carbon and total nitrogen was measured by a CHN analyser (CHN-O-Rapid, Foss-Heraeus, Germany).

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We chose three pairs of Vicia faba genotypes of small, medium and large seed sizes on the basis of earlier crude protein estimations (Genebank Gatersleben) in a way that within each pair one genotype has a low protein content (LP, 23 – 25 %) and the other one a high protein content (HP, 30 – 32 %). After growing the plants in the greenhouse the mature seeds were analysed for starch and extractable proteins. Thereby, similar significant differences in protein contents could be confirmed between the genotypes of each pair. On the other hand, the starch content was not statistically different although there was a trend to lower levels in the protein rich seeds (data not shown). Further experiments showed that the high protein seeds possessed a generally higher ratio of albumins to globulins and that the differences in protein contents became obvious only during the later stages of seed development (data not shown). We then analysed the total content of nitrogen and carbon in the seeds of these genotypes (Fig. 1). Total nitrogen was significantly higher in the seeds of all the three HP genotypes (13, 16 and 21 % for FAB 96, 13 and 148, respectively, P < 0.05). On the other hand, total amounts of carbon were not different. This indicated that the high protein genotypes could have mechanisms to specif-

PEPC enzyme assay Freshly harvested cotyledons were ground in 3 volumes (w/v) of 50 mmol/L HEPES, pH 7.4, 5 mmol/L MgCl2, 5 mmol/L dithiothreitol (DTT), 1 mmol/L EDTA, 1 mmol/L EGTA and 10 % glycerine. The assay was performed in 25 mmol/L TRIS, pH 8, 5 mmol/L MgCl2, 1 mmol/L KHCO3, 0.2 mmol/L NADH, 2 U malate dehydrogenase and 30 µL extract in a total volume of 0.5 mL and started with 2 or 5 mmol/L phosphoenolpyruvate for limiting or non-limiting conditions, respectively (Golombek et al. 1999).

Uptake and metabolism of 14C-glutamine, empty seed coat technique Intact cotyledons were transferred to (U-14C)glutamine solutions (5 MBq mmol/L –1; 60 mmol/L glutamine) in 25 mL conical flasks and incubated for 2 or 4 h on a shaker. Solutions contained 1 mmol/L CaCl2 buffered at pH 5.5 with 5 mmol/L MES/KOH and 100 mmol/L sucrose. The distribution of the 14C-label among starch, sugars and proteins was performed as described in Weber et al. 1998 b. The distribution of the 14C-label among the amino acids was analysed by thin-layer chromatography with crystalline cellulose (thickness 100 µm). Amino acids in the cationic fraction were separated in the first dimension with n-butanol, acetone, diethylamine and distilled water (60 : 60 : 12 : 30) and in the second dimension with isopropanol, formic acid and distilled water (60 : 3 : 13). Subsequently the 14C activity in individual amino acids was measured by autoradiography. Amino acids delivered from the seed coats were determined using the empty seed coat technique (Wolswinkel and Ammerlaan 1983).

Figure 1. Analysis of total nitrogen and carbon in mature embryos of three pairs of Vicia faba genotypes of small, medium and large seed sizes. From each pair one genotype has been characterized as having low (LP) and the other one as having high (HP) protein content. Plant material was dried at 80 ˚C to a constant weight and pulverised using a ball-mill. The tissue concentration of total carbon and total nitrogen was measured by a CHN analyser (CHN-O-Rapid, Foss-Heraeus, Germany).

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ically accumulate more nitrogen. In the following experiments we concentrated on the genotypes FAB 13 and FAB 154 (crude protein content, 30.1 and 23.9 %, respectively). Both are of medium seed size.

Contents of amino acids within developing seeds Synthesis of storage proteins is regulated at different levels. One important level is the availability of assimilates. To study potential differences in the levels of precursors for protein synthesis we analyzed the contents of free amino acids in the cotyledons of both the HP and the LP genotypes (FAB 13 and FAB 154) at three developmental stages (Fig. 2). Amounts were higher in cotyledons of the LP genotype during the early stages up to 0.5 g per seed. Thereafter, levels decreased in cotyledons of both genotypes and this was more pronounced in the LP genotype. In cotyledons larger than 1.5 g the HP genotype possessed a 2 to 3 fold higher amount. At this stage the differences in protein content occurred. This result indicated that a higher availability of amino acids could determine the high seed protein levels. In order to analyse possible differences in the unloading of free amino acids from the maternal seed tissue we used the empty seed coat technique (Wolswinkel and Ammerlaan 1983) to determine the relative composition of amino acids delivered from each type of seed coats. The coats of both genotypes mainly unloaded γ-aminobutyric acid (GABA), Ala, Gln, and Pro. During cotyledon development the amounts of GABA and Pro decreased whereas those of Ala and Gln increased (Fig. 3). There were only small differences in the composition of amino acids delivered from seed coat of either the HP or the LP genotype.

Uptake and partitioning of 14C-glutamine Isolated cotyledons of the HP and the LP genotypes were incubated with 60 mmol/L of 14C labeled glutamine. After two

Figure 3. Relative composition of amino acids delivered from each type of seed coats of FAB 13 (HP) and FAB 154 (LP) determined by the «empty seed coat technique».

Figure 4. Uptake rate of 14C-glutamine. Isolated cotyledons of the genotypes FAB 13 (HP) and FAB 154 (LP) were incubated with 60 mmol/L of 14 C labeled glutamine. After two and four hours cotyledons were removed and uptake rates were determined.

and four hours cotyledons were removed and uptake rates were determined. After the first two hours the uptake rate was around 4 to 4.5 µmol 14C glutamine g –1 h –1 and no significant differences occurred between the two genotypes (Fig. 4). However, after a longer incubation time of four hours, uptake rates for the HP cotyledons were significantly higher compared to the LP cotyledons (4 versus 1 µmol 14C Gln g –1 h –1). This indicates that the HP cotyledons could have a higher capacity for amino acid uptake.

Metabolism of 14C-glutamine

Figure 2. Contents of free amino acids in the cotyledons of both the high protein and the low protein genotypes (FAB 13 and FAB 154, respectively) at three developmental stages.

To study the further metabolization of 14C-glutamine the cotyledons were extracted after the incubation period and the labelled metabolites were analysed. 14C-glutamine was metabolised very fast. A pulse-chase experiment revealed that after two hours chase period more than 75 % was already metabolised (data not shown). After four hours period of 14C incubation the sum of the 14C metabolites was higher in cotyledons of the HP genotype (Fig. 5 A). The highest level of 14C label was found in the glutamate fraction indicating that glu-

Control of seed storage protein accumulation

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Figure 5. Metabolism of 14C-glutamine. Isolated cotyledons of the genotypes FAB 13 (HP) and FAB 154 (LP) were incubated with 60 mmol/L of 14C labeled glutamine (5 MBq mmol/L –1). (A) Distribution of 14C after supplying a 2 and 4 h pulse. (B) The relative 14C label incorporation into amino acids. Data are means of 3 independent experiments.

tamine is very rapidly converted into glutamate within the cotyledons. However, the cotyledons of the HP and LP genotypes, FAB 13 and FAB 154, did not differ in this respect. The activity of glutamate synthase was measured and again was not different (data not shown). Around 10 % of the 14C label was incorporated into GABA and another 5 % into an unknown amino acid which could not be identified yet. A considerable amount was also detected in the anionic fraction containing mainly organic acids (Fig. 5 A). The partitioning experiments further revealed that carbon from 14C-glutamine was only to a very minor extent incorporated into starch and only 3 % of the label appeared in the protein fraction after four hours of incubation (Fig. 5 A). However, after longer chase periods, the amounts of label found in the protein fraction increased (data not shown). Fig. 5 B shows the relative 14C label incorporation into amino acids on a percentage level. Especially the larger cotyledons of the HP genotype had a significant lower ratio of 14 C-glutamate to 14C-glutamine (Fig. 6). This could reflect the higher uptake rate of 14C-glutamine into the HP cotyledons rather than a different metabolisation of 14C-glutamine.

Phosphoenolpyruvate carboxylase activity is higher in cotyledons of the rich protein genotype Results from our experiments (Golombek et al. 1999) and those from others (Perez et al. 1993) allow to speculate that

Figure 6. Metabolism of 14C glutamine. Isolated cotyledons were incubated as in Figure 5 and the relative 14C label incorporation into amino acids glutamine and glutamate was determined.

differences in the partitioning of assimilates into the different storage product classes could account for a higher protein content. In this respect phosphoenolpyruvate carboxylase (PEPC) could play an important role. The enzyme catalyses the formation of oxalacetate from phosphoenolpyruvate and thereby replenishes the TCA-cycle which provides carbon skeletons for transamination reactions. We therefore analysed the maximum activity and activation status of PEPC in the cotyledons of the HP and the LP genotypes. In Fig. 7 it is shown that activity is similar in cotyledons up to 0.4 g but is

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Figure 7. PEP-carboxylase activity in cotyledons of the genotypes FAB 13 (HP) and FAB 154 (LP). Data are means of 3 independent experiments.

Table 1. AGP enzyme activity, starch and extractable proteins in mature cotyledons expressing AGP in antisense orientation. Three different homozygous lines as1, as12 and as19 and wildtype (wt) were analysed. Values in parentheses represent % of wt. Seeds were harvested from the same batch of plants. line

ADP glucose pyrophosphorylase (µmol g – 1 min – 1)

starch (mg g – 1)

as1 as12 as19 wildtype

0.13 ± 0.02 0.23 ± 0.01 0.39 ± 0.13 2.32 ± 0.5

271 ± 36 339 ± 34 309 ± 49 413 ± 19

(5.6) (9.9) (15.6) (100)

protein (mg g – 1)

(65.6) (82.1) (74.8) (100)

211 ± 16 191 ± 13 214 ± 11 172 ± 8

(122.7) (111.0) (124.4) (100)

significantly higher at later stages. There was no difference in PEPC activation stage between the seeds of the two genotypes (data not shown).

Decreasing starch by antisense-inhibition of ADPglucose pyrophosphorylase (AGP) led to higher protein content To answer the question whether a specific downregulation of one storage product causes a compensatory change in the other we made use of transgenic V. narbonensis seeds which seed-specifically expresses AGP in antisense orientation and therefore exhibits a block in starch biosynthesis. AGP-antisense inhibition led to characteristic compositional changes in mature cotyledons. Starch was only moderately decreased and the seeds accumulated more proteins (Table 1). In addition, cotyledonary development was substantially altered. Transgenic seeds contained more sugars and water and had a longer seed-filling phase (Weber et al. 2000). Partitioning experiments after feeding 14C-sucrose showed that the per-

Discussion In this work we attempted to analyze mechanisms which control protein accumulation in legume seeds. To this end V. faba genotypes of contrasting seed protein content were chosen from the Genebank, IPK Gatersleben. We compared various aspects of seed metabolism to answer the question whether the high protein seeds differ in respect to cotyledonary amino acid uptake and/or metabolism. Most of the analyses have been performed on two genotypes which differ by around 26 % in crude seed protein content (FAB 13, HP and FAB 154, LP) but are of similar seed weight. Quantitative analyses of the two main storage product classes, starch and proteins, as well as of total carbon and nitrogen content in mature seeds confirmed the expected changes in protein content and total nitrogen. However, starch and total carbon are not largely altered. We conclude therefore that both biosynthetic pathways are largely independent from each other. Similar conclusions have been made for soybean (Hayati et al. 1996), maize (Barneix et al. 1992) and wheat (Jenner et al. 1991). The higher seed protein content is realized only late in development and could be due to the capability of the HP genotype to maintain higher levels of protein synthesis for a longer time period. This result fits to the higher content of free amino acids present in the HP cotyledons during that stage. Because storage protein synthesis is highly regulated by the supply of nutrients (Balconi 1992, Müller and Knudsen 1993) the availability of free amino acids could be an important prerequisite to accumulate more protein. Barratt (1982) compared V. faba cultivars and found significant correlations between protein and free amino acids. Higher levels of amino acids could arise from increased supply from the mother plant (Lohaus et al. 1998) and/or from a more effective uptake into the cotyledons. Using the empty seed coat technique we found at least no significant differences in the relative proportion of amino acids unloaded from the seed coats of the HP and LP genotypes. However, this method is not suitable to analyse any possible quantitative variability. Most interestingly, the 14C glutamine uptake rates of the HP cotyledons are higher after a four hours period of incubation

Control of seed storage protein accumulation (Fig. 4) indicating a higher capacity to take up amino acids. In accordance, the sum of 14C metabolites is also higher in the HP seeds (Fig. 5 A). In addition, the higher proportion of 14 C-glutamine to 14C-glutamate (Fig. 6) can be expected when uptake rate rather than metabolism is increased. These results provide evidence for a more active amino acid uptake mechanism operating in the HP seeds which, particularly during the later stages of seed development, may be crucial for the higher seed protein content. Bennett and Spanswick (1983) reported that in soybean a saturable amino acid uptake system became increasingly important during later seed development. However, it is yet unknown whether amino acid transporters really play a rate limiting role in legume seeds (Tegeder et al. 2000, Miranda et al., in preparation). We have no evidence for differences in the metabolism of 14 C glutamine between seeds of the two genotypes. Glutamine is found to be rapidly converted into glutamate (Fig. 5), probably by glutamate synthase. A significant proportion of the glutamate is thereafter converted to GABA by glutamate decarboxylase. High amounts of GABA were also measured in developing soybean seeds (Tuin and Shelp 1996). GABA may play an important role in the amino acid metabolism of seeds. This metabolite can be converted into succinate thereby providing a link between glutamine and the Krebs cycle. Increased organic acids from the Krebs cycle will then be available for amination reactions (Tuin and Shelp 1996, Murray and Cordoba-Edvards 1984). A major problem comparing genotypes differing in seed protein content may arise when these are not isogenic and therefore most probably differ for a range of other properties than seed protein content. For example, the FAB 13 and FAB 154 genotypes, we used in this study, slightly differ in final seed size and seed fill duration (Golombek and Weber unpublished results). One way to circumvent such problems is to use transgenic plants in which the activity of only one enzyme is altered. V. narbonensis plants expressing seedspecifically a AGP-cDNA in antisense orientation could represent a suitable model. The transgenic alterations in the mid to late stages of embryo development in V. narbonensis lead to a slight decrease of starch and increase of sucrose and water content from this stage on, and in mature seeds, to increased fresh weight but unaltered dry weight, and increased protein (Weber et al. 2000). More recent experiments revealed that a reduced carbon flux into starch does not necessarily lead to increased C partitioning into storage proteins (Rolletschek et al., in preparation). Moreover, the block in starch synthesis can have pleiotropic effects on water content and induces temporal changes in seed development. For example, a resulting longer seed fill duration could partially explain the observed elevated protein content in AGPantisense seeds (Weber et al. 2000). Acknowledgements. We thank Elsa Fessel, Angela Schwarz and Katrin Blaschek for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft, SPP3221005 and

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SFB 363. U.W. acknowledges additional support by the Fonds der Chemischen Industrie.

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