Pattern analysis suggests that phosphoenolpyruvate carboxylase in maturing soybean seeds promotes the accumulation of protein

ABSTRACT

The protein and oil contents in soybean seeds are major factors in seed quality. Seed proteins and oils are synthesized from sucrose and nitrogenous compounds transported into maturing seeds. In this study, we compared changes in the activity of phosphoenolpyruvate carboxylase (PEPC) and the accumulation profiles of protein and oil in maturing seeds of two soybean cultivars, which exhibit different protein and oil contents in seeds, to determine the interrelationships of them. A principal component analysis indicated a concordance of seed PEPC activity with the protein content, but did not with the oil content. PEPC activity per seed was highest in the late maturation stage, when the physiological status of the vegetative organs drastically changed. The high-protein cultivar had higher PEPC activity compared to the low-protein cultivar. These results highlight the biological role of PEPC in the synthesis of protein, therefore it was implied that PEPC could be a biomarker in soybean breeding.

Abbreviations: ANOVA: analysis of variance; DS: developmental stage; DW: dry weight; FW: fresh weight; NIR: near infrared; PEP(C): phosphoenolpyruvate (carboxylase); PC(A): principal component (analysis); S.E.: standard error; WC: water content.     

Characterisation of seeds of a C4 phosphoenolpyruvate carboxylase-deficient mutant of Amaranthus edulis

Seeds from the C(4) plant Amaranthus edulis were studied as part of the characterisation of a mutant (designated LaC(4) 2.16), which contains reduced amounts (5% of wild type) of the photosynthetic leaf form of phosphoenolpyruvate carboxylase (PEPC). On a per seed basis, the amount of PEPC activity was not significantly altered, while the weight and protein content of the mutant seeds were 34% lower than that of the wild type. Western gel blot analysis detected two PEPC polypeptides with molecular masses of 105 kDa (minor) and 100 kDa (major). The determination of in vitro phosphorylation in reconstituted assays revealed the presence of both calcium-dependent and calcium-independent PEPC-kinase activities in protein extracts of wild-type and mutant seeds. However, PEPC proteins were phosphorylated in dry seeds, and PEPC phosphorylation did not occur in vivo during seed imbibition in the presence of (32) P-phosphate. In contrast, (32) P-phosphate was incorporated into a range of proteins in wild-type seeds, but not in mutant seeds. In addition, ATP content was much reduced in germinating mutant seeds and this did not increase following the supply of phosphate. Collectively, these data suggest that the deficiency in C(4) PEPC in mutant A. edulis leaves has no effect on C(3) -type PEPC content and phosphorylation state in seeds, but causes impairment of energy production, thereby accounting for the reduced germination of the mutant.     

Ectopic expression of an amino acid transporter (VfAAP1) in seeds of Vicia narbonensis and pea increases storage proteins

Storage protein synthesis is dependent on available nitrogen in the seed, which may be controlled by amino acid import via specific transporters. To analyze their rate-limiting role for seed protein synthesis, a Vicia faba amino acid permease, VfAAP1, has been ectopically expressed in pea (Pisum sativum) and Vicia narbonensis seeds under the control of the legumin B4 promoter. In mature seeds, starch is unchanged but total nitrogen is 10% to 25% higher, which affects mainly globulin, vicilin, and legumin, rather than albumin synthesis. Transgenic seeds in vitro take up more [14C]-glutamine, indicating increased sink strength for amino acids. In addition, more [14C] is partitioned into proteins. Levels of total free amino acids in growing seeds are unchanged but with a shift toward higher relative abundance of asparagine, aspartate, glutamine, and glutamate. Hexoses are decreased, whereas metabolites of glycolysis and the tricarboxylic acid cycle are unchanged or slightly lower. Phosphoenolpyruvate carboxylase activity and the phosphoenolpyruvate carboxylase-to-pyruvate kinase ratios are higher in seeds of one and three lines, indicating increased anaplerotic fluxes. Increases of individual seed size by 20% to 30% and of vegetative biomass indicate growth responses probably due to improved nitrogen status. However, seed yield per plant was not altered. Root application of [15N] ammonia results in significantly higher label in transgenic seeds, as well as in stems and pods, and indicates stimulation of nitrogen root uptake. In summary, VfAAP1 expression increases seed sink strength for nitrogen, improves plant nitrogen status, and leads to higher seed protein. We conclude that seed protein synthesis is nitrogen limited and that seed uptake activity for nitrogen is rate limiting for storage protein synthesis.     

Uptake and allocation of carbon and nitrogen in Vicia narbonensis plants with increased seed sink strength achieved by seed-specific expression of an amino acid permease

Over-expressing an amino acid permease in Vicia narbonensis seeds increases sink strength for N that is evident from the higher seed protein content and seed weight. Here, the effect of increased seed sink strength of line AAP-12 on growth, development, and on whole plant carbon and nitrogen uptake and partitioning is analysed. AAP-12 plants have a prolonged growth period. Accumulation and partitioning of dry matter and N in leaves, stems, and pods are higher whereas remobilization to the seeds is delayed, indicating that the switch from growth to reserve allocation and remobilization is delayed. Measuring uptake and allocation of (15)N-ammonia applied via the roots revealed a higher and longer label uptake period during maturation. Measuring whole plant carbon fixation and allocation after (13)C labelling shows higher levels at maturation, particularly in seeds, indicating higher seed sink strength for C and increased allocation into maturing seeds. Levels of cytokinins were dramatically increased in AAP-12 seeds indicating its role in nitrogen-mediated growth stimulation. AAP-12 seeds have higher natural abundances for (13)C indicating increased C fixation via PEP carboxylase in order to meet the higher demand of carbon acceptors for amino acid synthesis. In summary, increased seed sink strength for N in AAP-12 stimulates seed growth, but also that of vegetative organs, which finally leads to a higher ratio of vegetative to seed biomass at maturity and thus a lower harvest index. Therefore, the increased N uptake due to higher seed demand of AAP-12 is partly compensated by growth stimulation of vegetative organs.     

花生种子的发育与贮藏蛋白质的合成和积累

花生果针入土后16天(16 DAP),种子干重和鲜重开始迅速增加。整个发育阶段可分为5个时期:组织分化期(0～20 DAP)、成熟前期(21～28 DAP)、成熟中期(29～40DAP)、成熟中后期(41～62 DAP)和成熟后期(63～88DAP)。种子发芽率在成熟前期和中期迅速提高并到达最大值,而苗成活率在成熟中后期达到最大值,苗鲜重则以88 DAP种子的为最大。种子发育过程中,贮藏蛋白质的合成与积累模式与种子干重变化相似。SDS-PAGE分析表明,种子发育初期(16 DAP)子叶中已积累花生球蛋白和伴花生球蛋白I。双向凝胶电泳显示花生球蛋白各个亚基在20DAP时均已存在,伴花生球蛋白I的主要亚基在整个发育过程中其等电点有所变化,含量也逐渐增加。其他蛋白质在种子发芽力形成阶段(20～40 DAP)的变化较为显著。     

AAP1 regulates import of amino acids into developing Arabidopsis embryos

The embryo of Arabidopsis seeds is symplasmically isolated from the surrounding seed coat and endosperm, and uptake of nutrients from the seed apoplast is required for embryo growth and storage reserve accumulation. With the aim of understanding the importance of nitrogen (N) uptake into developing embryos, we analysed two mutants of AAP1 (At1g58360), an amino acid transporter that was localized to Arabidopsis embryos. In mature and desiccated aap1 seeds the total N and carbon content was reduced while the total free amino acid levels were strongly increased. Separately analysed embryos and seed coats/endosperm of mature seeds showed that the elevated amounts in amino acids were caused by an accumulation in the seed coat/endosperm, demonstrating that a decrease in uptake of amino acids by the aap1 embryo affects the N pool in the seed coat/endosperm. Also, the number of protein bodies was increased in the aap1 endosperm, suggesting that the accumulation of free amino acids triggered protein synthesis. Analysis of seed storage compounds revealed that the total fatty acid content was unchanged in aap1 seeds, but storage protein levels were decreased. Expression analysis of genes of seed N transport, metabolism and storage was in agreement with the biochemical data. In addition, seed weight, as well as total silique and seed number, was reduced in the mutants. Together, these results demonstrate that seed protein synthesis and seed weight is dependent on N availability and that AAP1-mediated uptake of amino acids by the embryo is important for storage protein synthesis and seed yield.     

Regulation of phosphoenolpyruvate carboxylase phosphorylation by metabolites and abscisic acid during the development and germination of barley seeds

During barley (Hordeum vulgare) seed development, phosphoenolpyruvate carboxylase (PEPC) activity increased and PEPC-specific antibodies revealed housekeeping (103-kD) and inducible (108-kD) subunits. Bacterial-type PEPC fragments were immunologically detected in denatured protein extracts from dry and imbibed conditions; however, on nondenaturing gels, the activity of the recently reported octameric PEPC (in castor [Ricinus communis] oil seeds) was not detected. The phosphorylation state of the PEPC, as judged by l-malate 50% inhibition of initial activity values, phosphoprotein chromatography, and immunodetection of the phosphorylated N terminus, was found to be high between 8 and 18 d postanthesis (DPA) and during imbibition. In contrast, the enzyme appeared to be in a low phosphorylation state from 20 DPA up to dry seed. The time course of 32/36-kD, Ca(2+)-independent PEPC kinase activity exhibited a substantial increase after 30 DPA that did not coincide with the PEPC phosphorylation profile. This kinase was found to be inhibited by l-malate and not by putative protein inhibitors, and the PEPC phosphorylation status correlated with high glucose-6-phosphate to malate ratios, thereby suggesting an in vivo metabolic control of the kinase. PEPC phosphorylation was also regulated by photosynthate supply at 11 DPA. In addition, when fed exogenously to imbibing seeds, abscisic acid significantly increased PEPC kinase activity. This was further enhanced by the cytosolic protein synthesis inhibitor cycloheximide but blocked by protease inhibitors, thereby suggesting that the phytohormone acts on the stability of the kinase. We propose that a similar abscisic acid-dependent effect may contribute to produce the increase in PEPC kinase activity during desiccation stages.     

Accumulation of a Brazil nut albumin in seeds of transgenic canola results in enhanced levels of seed protein methionine

We have increased the methionine content of the seed proteins of a commercial winter variety of canola by expressing a chimeric gene encoding a methionine-rich seed protein from Brazil nut in the seeds of transgenic plants. Transgenic canola seeds accumulate the heterologous methionine-rich protein at levels which range from 1.7% to 4.0% of the total seed protein and contain up to 33% more methionine. The precursor of the methionine-rich protein is processed correctly in the seeds, resulting in the appearance of the mature protein in the 2S protein fraction. The 2S methionine-rich protein accumulates in the transgenic seeds at the same time in development as the canola 11S seed proteins and disappears rapidly upon germination of the seed. The increase in methionine in the canola seed proteins should increase the value of canola meal which is used in animal feed formulations.     

Seed preference and energy intake of Goldfinches Carduelis carduelis in the breeding season

The food of a population of Europeans Goldfinches breeding in Orchards was studied. More than 5% of the total foodstuff consumed was met by the seeds of seven food plants. Water, lipid, carbohydrate and protein content was determined from 20 different plants which Goldfinches used during the breeding season in order to calculate energy content. The energy of fresh seeds varied from 1.88 kJ/g in Tussilago farfara to 12.14 kJ/g in Dactylis glomerata. The dependence of the rate of energy intake (profitability) on the pecking rate and the size (weight) of the seeds was calculated. The profitability of various seeds is in agreement with the relative number of birds using them. Calculation of the feeding time that a Goldfinch needs during daylight hours, if it feeds only on a particular plant, indicates that there are several foods that cannot fill the energy demands of the bird during the breeding season. These are eaten only if others with a high energy content are not available. Thus food selection in Goldfinches depends, apart from availability, on seed profitability. The oxygen consumption of a male Goldfinch was significantly higher when the animal was feeding on seed heads of Tussilago farfara and Taraxacum officinale than when it fed on loose dry seeds, and so, different feeding techniques have to be taken into account in calculating net energy gain.     

Protein and oil concentration of soybean seed cultured in vitro using nutrient solutions of differing glutamine concentration

Oil and protein are the most valuable components of soybean seed. Evidence indicates that growth and composition of soybean seed are controlled by supplies of carbon and nitrogen provided by the maternal plant to the seed, but it is difficult experimentally to control and quantify the precise amount of carbon and nitrogen provided to the seed by the whole plant. To examine whether oil and protein concentrations are affected by the supply of nitrogen to the seed, immature soybean seeds (Glycine max cv. Williams 82) were grown in vitro in nutrient solutions containing 20, 40, 60 or 80 mM of glutamine. The seeds were incubated in Erlenmeyer flasks for 8 days at 25°C. The rate of dry matter accumulation changed from 7.2 to 8.3 mg seed⁻¹ day⁻¹ as the glutamine concentration increased from 20 to 80 mM but the differences were not significant (P 0.05). Seed protein concentration increased as glutamine concentration increased from 294 mg g⁻¹ at 20 mM glutamine to as high as 445 mg g⁻¹ at 80 mM glutamine. Typical in vivo protein concentration of mature soybean seeds is about 400 mg g⁻¹. Oil and protein concentrations were negatively correlated (r²= 0.44), which indicates that oil and protein synthesis are interrelated. Protein synthesis was favoured over oil synthesis when nitrogen became more abundant. The seeds used in this study clearly demonstrated a capacity to respond to nitrogen availability with changes in seed protein concentration.     

Regulation of storage lipid metabolism in developing and germinating lupin (<Emphasis Type="Italic">Lupinus</Emphasis> spp.) seeds

The main storage compound in lupin seeds is protein, whose content can reach up to 45–50 % of dry matter. However, seeds of some lupin species can also contain quite a large amount of storage lipid. The range of lipid content in lupin seeds is from about 6 to about 20 % of dry matter. Storage lipid in developing seeds is synthesized mainly from sugars delivered by mother plants. During seed germination, one of the main end-products of storage lipid breakdown is also sugars. Thus, the sugar level in tissues is considered an important regulatory agent, during both lipid accumulation and lipid breakdown. Generally, in developing legume seeds, there is a strong negative relation between accumulation of storage protein and storage lipid. Results obtained in developing lupin cotyledons cultured in vitro pointed to the possibility of a positive relation between protein and lipid accumulation. Such a positive effect could be caused by nitrate. During lupin seed germination and seedling development, the utilization of storage lipid is enhanced under sugar deficiency conditions in tissues and is controlled at the gene expression level. However, under sugar starvation conditions, autophagy is significantly enhanced, and it can cause disturbances in storage lipid breakdown. The hypothesis of pexophagy, i.e., autophagic degradation of peroxisomes under sugar starvation conditions during lupin seed germination, has been taken into consideration. The flow of lipid-derived carbon skeletons to amino acids was discovered in germinating lupin seeds, and this process is clearly more intense in sucrose-fed embryo axes. At least four alternative or mutually complementary pathways of carbon flow from storage lipid to amino acids in germinating lupin seeds are postulated. The different strategies of storage compound breakdown during lupin seed germination are also discussed.     

Bioenergetic considerations in the improvement of oil content and quality in oil-seed crops

Summary Production values (PVs), defined as the weight of the end product/weight of the substrate required for carbon skeletons and energy production, were calculated for plant fatty acids. The PVs varied from 0.361 to 0.300 with linolenic acid having the lowest value. In general, the PVs of unsaturated fatty acids were lower than those of saturated fatty acids of similar chain lengths. Using this basic information, PVs of (A) oils from different oilseed crops, based on their standard fatty acid composition and (B) seed biomass with specified oil content and fatty acid composition were calculated. 1/PV gives the glucose required for the biosynthesis of 1 g end product and thus an estimate of the photosynthate requirement for the desired breeding goal can be estimated. Such calculations show that increasing oil percentage in seeds has a maximum energy cost when the increase in oil is associated with a decrease in the amount of carbohydrates where there is no change in protein concentration. Reduction of erucic acid content in the rapeseed oil did not alter its PV. It is inferred that there are no serious bioenergetic constraints in altering the fatty acid composition.     

Overexpression of phosphoenolpyruvate carboxylase from Jatropha curcas increases fatty acid accumulation in Nicotiana tabacum

Jatropha curcas L. is an excellent biofuel crop, which displays a high efficiency of carbon absorption, and seed oil of Jatropha can be efficiently processed to produce high-quality biodiesel. Plant phosphoenolpyruvate carboxylases (PEPCs) play important roles not only in initial fixation of atmospheric CO₂ in C₄ and Crassulacean acid metabolism (CAM) plants, but also in fatty acid biosynthesis in seeds of oil plants by regulating carbon partitioning. Here, we identified JcPEPC1 from J. curcas L. by homology cloning, and alignment analysis of protein sequence revealed JcPEPC1 was a plant C₃-type PEPC, and shared high similarity to PEPC of castor oil plant Ricinus communis. We implemented detailed functional characterization of JcPEPC1 by expression analysis and transgenic tobacco. JcPEPC1 gene expressed in the leaves and seeds of J. curcas L., and remarkable increase of expression level was also detected at seed oil-accumulating stages. We overexpressed JcPEPC1 in tobacco, and showed the enzymatic activity of PEPC in transgenic plants was notably higher than wild type. Gas chromatography (GC) analysis elucidated the composition and total content of fatty acids were also altered. This study indicated JcPEPC1 played a fundamental role in fatty acid biosynthesis in Jatropha seeds. Our results proposed enhanced PEPC activity of Jatropha could improve biosynthesis of fatty acid, which implied critical functions in primary metabolism of non-photosynthetic PEPC.     

Improvement of the nutritional quality of legume seed storage proteins by molecular breeding

Legume seeds contain a large amount of proteins and are one of the essential protein sources for humans and animals. However, the protein, in legume seeds is usually poor in sulfur-containing amino acids, and its nutritional value is lower than the protein from animal sources. Recently plant breeding has become available by the introduction of molecular biology, and a technique, called molecular breeding, was applied to the production of legume seeds that contain proteins with high nutritional quality. This review describes the expression of legume seed protein genes and the transformation of legume plants. Approaches to improve the legume seed storage protein will be discussed.     

Physiological and metabolic enzymes activity changes in transgenic rice plants with increased phosphoenolpyruvate carboxylase activity during the flowering stage

To compare the differences in physiology and metabolism between phosphoenolpyruvate carboxylase (PEPC) transgenic rice and its control, untransformed wild rice, dry matter accumulation, soluble sugar, starch and protein contents and enzyme activities were determined in different plant parts during flowering. Results revealed that PEPC transgenic rice had higher dry weights for leaf, stem and sheath as well as panicle than the untransformed wild rice did, with the largest increase in the panicle. Soluble sugar and protein content in the grains of PEPC transgenic rice were significantly enhanced while starch content changed less. PEPC transgenic rice exhibited high levels of PEPC activity, manifesting in high net photosynthetic rates during flowering. Moreover, transgenic rice with high PEPC expression levels also had elevated levels of the enzymes such as sucrose-p-synthase and sucrose synthase, which may confer a higher capacity to assimilate CO₂ into sucrose. Little increase in grain starch content was observed in transgenic plants due to the stable activities of starch synthase and Q enzyme. However, the PEPC transgenic rice plant induced the activities of nitrate reductase, glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, glutamine synthetase, and asparagine synthase to high levels, as compared with the untransformed rice plant. PEPC activity was correlated with protein content in grains and the enzymes of nitrogen metabolism, suggesting that high PEPC activity in transgenic rice might be able to redirect carbon and nitrogen flow by regulating some enzymes related to carbon or nitrogen metabolisms. These results may help to understand how the C₃ plants possessing a C₄-like photosynthesis pathway worked by expression of PEPC.     

Seed dimorphism in Salicornia europaea: Nutrient reserves

Median and lateral seeds of Salicornia europaea L. were separately analysed for their sizes and nutrient reserves. The mean air-dry weight of a single median and lateral seed was 0.31 and 0.25 mg, respectively. The composition as well as the concentration of the nutrient reserves were similar in both seed types. The bulk of the cations was derived from K⁺, followed by Mg²⁺, Na⁺ and Ca²⁺. The chloride content was somewhat higher than the sodium content, and phosphate was equalled by acid soluble Ca²⁺ and Mg²⁺. Starchy compounds and sucrose were present in equal amounts, each of them accounted for about 50% of the carbohydrates. Glucose and fructose were less than 1%. Protein-nitrogen (ethanol-insoluble N) was about 34 g (kg dry seeds)⁻¹. About 7 g (kg dry seeds)⁻¹ was ethanol-soluble nitrogen, of which 10% was derived from amino acids. The total lipid content was more than 290 g (kg dry seeds)⁻¹, 65% were calculated to be glycerides. More than 90% of the fatty acids consisted of linoleic and oleic acids, the majority (72%) of which was linoleic acid.     

In Vivo and in Vitro Phosphorylation of the Phosphoenolpyruvate Carboxylase from Wheat Seeds during Germination

Phosphoenolpyruvate carboxylase (PEPC) activity was detected in the aleurone endosperm of wheat (Triticum aestivum cv Chinese Spring) seeds, and specific anti-Sorghum C4 PEPC polyclonal anti-bodies cross-reacted with 103- and 100-kD polypeptides present in dry seeds and seeds that had imbibed; in addition, a new, 108-kD polypeptide was detected 6 h after imbibition. The use of specific anti-phosphorylation-site immunoglobulin G (APS-IgG) identified the presence of a phosphorylation motif equivalent to that found in other plant PEPCs studied so far. The binding of this APS-IgG to the target protein promoted changes in the properties of seed PEPC similar to those produced by phosphorylation, as previously shown for the recombinant Sorghum leaf C4 PEPC. In desalted seed extracts, an endogenous PEPC kinase activity catalyzed a bona fide phosphorylation of the target protein, as deduced from the immunoinhibition of the in vitro phosphorylation reaction by the APS- IgG. In addition, the major, 103-kD PEPC polypeptide was also shown to be radiolabeled in situ 48 h after imbibition in [32P]orthophosphate. The ratio between optimal (pH 8) and suboptimal (pH 7.3 or 7.1) PEPC activity decreased during germination, thereby suggesting a change in catalytic rate related to an in vivo phosphorylation process. These collective data document that the components needed for the regulatory phosphorylation of PEPC are present and functional during germination of wheat seeds.