Gluconeogenesis from amino acids in germinating castor bean endosperm and its role in transport to the embryo

During germination of the castor bean all of the contents of the endosperm are ultimately transported to the embryo through the cotyledon or respired. A net loss of nitrogen from the endosperm begins about the fourth day, i.e. at the time when embryo growth and fat breakdown are also beginning. Amino acid analysis of the exudate from the cotyledons, still enclosed in the endosperm, showed that the amounts of aspartate, glutamate, glycine, and alanine were very low and that glutamine made up 40% of the amino acids in the exudate.

Amino acids labeled with ¹⁴C were applied to intact excised endosperms to follow utilization. Aspartate, glutamate, alanine, glycine, serine, and leucine were converted to sugar to varying extents. Proline, arginine, valine, and phenylalanine were not appreciably converted to sugars. Proline and glutamate were converted to glutamine. When ¹⁴C-glutamate, aspartate, and alanine were added to the outer endosperm of intact seedlings, only sugars and glutamine contained appreciable label in the exudate. When ¹⁴C-valine was added, it was virtually the only labeled compound in the exudate.

The results show that amino acids which on deamination can give rise to intermediates in the pathway of conversion of fat to sucrose are largely converted to sucrose and the nitrogen transported as glutamine. Other amino acids released from the endosperm protein are transported intact into the seedling axis. Some carbon from the gluconeogenic amino acids is also transported as glutamine.

     

Metabolism of C-Maltose in Avena fatua Seeds During Germination

Non-dormant and dormant seeds of Avena fatua metabolize ¹⁴C-maltose in different ways: in non-dormant seeds, ¹⁴C-maltose administered to the endosperm is readily converted to sucrose in the scutellum and translocated to the embryo; in dormant seeds, little sucrose is synthesized from ¹⁴C-maltose, and maltose and glucose tend to accumulate in the endosperm. It is suggested that biosynthesis of sucrose is essential for effective transport of the endosperm reserve to the embryonic axis in germinating seeds.     

Cessation of assimilate uptake in maturing soybean seeds

In vitro assimilate uptake and metabolism were evaluated in embryos of known age isolated from seeds at mid-podfilling through physiological maturity. The capacity of isolated Wye soybean embryos to take up exogenous [¹⁴C]sucrose dropped nearly 4-fold in less than 1 week at incipient cotyledon yellowing. This drop in rate of sucrose uptake coincided with cessation of seed growth as well as rapid decline in leaf photosynthetic rate that preceded leaf yellowing. Conversely, the rate of [³H]glutamine uptake by cotyledons increased as they yellowed. Yellow cotyledons also rapidly converted exogenous [³H]glutamine to ethanolinsoluble components, but converted little exogenous [¹⁴C]sucrose to ethanol-insoluble components, primarily because of greatly reduced sucrose uptake. Sustained import and metabolism of amino acids remobilized from senescing leaves may prolong seed growth beyond loss of photosynthetic competency and sucrose availability.     

Transport of purine and pyrimidine bases and nucleosides from endosperm to cotyledons in germinating castor bean seedlings

During germination and early growth of castor bean (Ricinus communis), all cellular constituents of the endosperm are eventually transferred to the growing embryo. The present results bear on the transport of breakdown products of nucleic acids. The total content of nucleic acids and nucleotides declines rapidly between day 4 and day 8 of seedling development. Concomitant with this decline, a secretion of adenosine, guanosine, and adenine from excised endosperms into the incubation medium takes place, accompanying a much more extensive release of sucrose and amino acids. Release of nucleotides could not be detected. The rates of release were linear for at least 5 hours for all compounds measured, indicating that they were liberated due to a coordinated metabolism. Uptake studies with cotyledons removed from the seedling showed that these have the ability to absorb all the substances released from the endosperm. Besides sucrose and amino acids, both nucleosides and free purine and pyrimidine bases were taken up by the cotyledons with high efficiency. AMP was also transported whereas ATP was not. Kinetic analyses were carried out to estimate the maximal uptake capacities of the cotyledons. Rates of uptake were linear for at least 1 to 2 hours and saturation kinetics were observed for all substances investigated. It is concluded that nucleosides can serve best as transport metabolites of nucleic acids, inasmuch as they are taken up by the cotyledons with the highest efficiency, the V_max/K_m ratios being considerably higher than those found for free purine and pyrimidine bases. For both adenosine and adenine transport, the V_max was about 2 micromoles per hour per gram fresh weight, and the K_m values were 0.12 and 0.37 millimolar, respectively. The rates of metabolite release from the endosperm and the capacity of the absorption system in the cotyledons are shown to account for the observed rates of disappearance of nucleic acids from the endosperm and efficient transport to the growing embryo.     

Concentrations of sucrose and nitrogenous compounds in the apoplast of developing soybean seed coats and embryos

The apoplast of developing soybean (Glycine max cv Hodgson) embryos and seed coats was analyzed for sucrose, amino acids, ureides, nitrate, and ammonia. The apoplast concentration of amino acids and nitrate peaked during the most rapid stage of seed filling and declined sharply as the seed attained its maximum dry weight. Amino acids and nitrate accounted for 80 to 95% of the total nitrogen, with allantoin and allantoic acid either absent or present in only very small amounts. Aspartate, asparagine, glutamate, glutamine, serine, alanine, and γ-aminobutyric acid were the major amino acids, accounting for over 70% of the total amino acids present. There was a nearly quantitative conversion of glutamine to glutamate between the seed coat and embryo, most likely resulting from the activity of glutamate synthase found to be present in the seed coat tissue. This processing of glutamine suggests a partly symplastic route for solutes moving from the site of phloem unloading in the seed coat to the embryo.     

The amino acid permease AAP8 is important for early seed development in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The development of seeds depends on the import of carbohydrates and amino acids supplied by the maternal tissue via the phloem. Several amino acid transporters have been reported to be expressed during seed and silique development in Arabidopsis thaliana (L.) Heynh. Here we show that mutants lacking the high affinity amino acid permease 8 (At1g10010) display a severe seed phenotype. The overall number of seeds and the number of normally developed seed is reduced by ∼50% in siliques of the Ataap8 T-DNA insertion mutant. This result could be reproduced in plants where expression of AtAAP8 is targeted with an RNAi approach. The seed phenotype is correlated with a specifically altered amino acid composition of young siliques. Aspartic acid and glutamic acid are significantly reduced in young siliques of the mutants. In correlation with the fact that AAP8 is a high affinity transporter for acidic amino acids, translocation of ¹⁴C-labelled aspartate fed via the root system to seeds of the mutants is reduced. AAP8 plays a crucial role for the uptake of amino acids into the endosperm and supplying the developing embryo with amino acids during early embryogenesis.     

Mobilization of Endosperm Reserves and Uptake of Sucrose and Valine by the Cotyledons of Euphorbia lathyris L.

Upon germination, the endosperm triacylglycerols and proteinswere converted to sucrose and amino acids. During early postgerminativegrowth, the rate of sucrose and amino acid production exceededthe rate of uptake by the cotyledons. As a result, the levelsof total amino acid and sucrose in the endosperm increased;maximum levels were reached at 7 d and 10 d after imbibition(DAI), respectively. Intact seedlings were used to measure thedevelopment of valine, arginine, glutamic acid, and sucroseuptake rate throughout the course of endosperm depletion. Maximumamino acid uptake rates were measured at around 9 DAI, the highestuptake rate for sucrose was obtained at 12 DAI (just beforedepletion of the endosperm). The daily increase of sucrose andamino acid uptake could be manipulated, by replacing the endospermwith a pre-incubation solution during 1 d. The increase in sucroseuptake in vitro was equal to that measured with intact seedlingswhen the cotyledons were pre-incubated in 10 mol m^–3 sucrose.Higher sucrose concentrations reduced the increase of sucroseuptake; at 300 mol m^–3 sucrose (corresponding to the meanendosperm sucrose concentration) sucrose uptake after pre-incubationwas even lower than before. This reduction was largely counteractedwhen the pre-incubation solution was supplemented with minerals.The development of the valine uptake was hardly affected bysucrose, but was inhibited by several amino acids. Key words: Euphorbia lathyris seedling, sucrose uptake, amino acid uptake, reserve mobilization     

Transport and Metabolism of a Sucrose Analog (1'-Fluorosucrose) into Zea mays L. Endosperm without Invertase Hydrolysis

1′-Fluorosucrose (FS), a sucrose analog resistant to hydrolysis by invertase, was transported from husk leaves into maize (Zea mays L., Pioneer Hybrid 3320) kernels with the same magnitude and kinetics as sucrose. ¹⁴C-Label from [¹⁴C]FS and [¹⁴C]sucrose in separate experiments was distributed similarly between the pedicel, endosperm, and embryo with time. FS passed through maternal tissue and was absorbed intact into the endosperm where it was metabolized and used in synthesis of sucrose and methanol-chloroform-water insolubles. Accumulation of [¹⁴C] sucrose from supplied [¹⁴C]glucosyl-FS indicated that the glucose moiety from the breakdown of sucrose (here FS), which normally occurs in the process of starch synthesis in maize endosperm, was available to the pool of substrates for resynthesis of sucrose. Uptake of FS into maize endosperm without hydrolysis suggests that despite the presence of invertase in maternal tissues and the hydrolysis of a large percentage of sucrose unloaded from the phloem, hexoses are not specifically needed for uptake into maize endosperm.     

Degree of pectin methyl esterification in endosperm cell walls is involved in embryo bending in Arabidopsis thaliana

The endosperm is a transitory structure involved in proper embryo elongation. The cell walls of mature seed endosperm are generally composed of a uniform distribution of cellulose, unesterified homogalacturonans, and arabinans. Recent studies suggest that changes in cell wall properties during endosperm development could be related to embryo growth. The degree of methyl esterification of homogalacturonans may be involved in this endosperm tissue remodelling. The relevance of the degree of homogalacturonan methyl esterification during seed development was determined by immunohistochemical analyses using a panel of probes with specificity for homogalaturonans with different degrees of methyl esterification. Low-esterified and un-esterified homogalacturonans were abundant in endosperm cells during embryo bending and were also detected in mature embryos. BIDXII (BDX) could be involved in seed development, because bdx-1 mutants had misshapen embryos. The methyl esterification pattern described for WT seeds was different during bdx-1 seed development; un-esterified homogalacturonans were scarcely present in the cell walls of endosperm in bending embryos and mature seeds. Our results suggested that the degree of methyl esterification of homogalacturonans in the endosperm cell wall may be involved in proper embryo development.     

A Cascade of Sequentially Expressed Sucrose Transporters in the Seed Coat and Endosperm Provides Nutrition for the Arabidopsis Embryo

Developing plant embryos depend on nutrition from maternal tissues via the seed coat and endosperm, but the mechanisms that supply nutrients to plant embryos have remained elusive. Sucrose, the major transport form of carbohydrate in plants, is delivered via the phloem to the maternal seed coat and then secreted from the seed coat to feed the embryo. Here, we show that seed filling in Arabidopsis thaliana requires the three sucrose transporters SWEET11, 12, and 15. SWEET11, 12, and 15 exhibit specific spatiotemporal expression patterns in developing seeds, but only a sweet11;12;15 triple mutant showed severe seed defects, which include retarded embryo development, reduced seed weight, and reduced starch and lipid content, causing a “wrinkled” seed phenotype. In sweet11;12;15 triple mutants, starch accumulated in the seed coat but not the embryo, implicating SWEET-mediated sucrose efflux in the transfer of sugars from seed coat to embryo. This cascade of sequentially expressed SWEETs provides the feeding pathway for the plant embryo, an important feature for yield potential.     

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.     

Movement of C-compounds from Maternal Tissue into Maize Seeds Grown in Vitro

Uptake from nutrient media into the cob and translocation of various ¹⁴C-compounds from maternal tissue (cob) into developing maize seeds was examined by using caryopsis cultures. Based on relative ¹⁴C concentrations in the cob and the endosperm, it was concluded that the relative efficiencies of movement of amino acids (leucine, phenylalanine, proline), vitamins (thiamine HCl, nicotinic acid), and nucleic acid bases (adenine, thymine) from the cob to the endosperm were 11 to 250 times lower than that of sucrose. Thiamine was unique in that it was concentrated in the embryo at a level that was almost 10 times higher than in the endosperm. The absence of auxotrophic mutants requiring an organic supplement in higher plants (other than thiamine auxotrophs) may be explained by inadequate translocation of these essential metabolites into the mutant zygotes (embryos) to enable their development to mature seeds.     

Endosperm protein of wheat seed as a determinant of seedling growth

Seed of a Mexican semidwarf wheat (Triticum aestivum L. cv. Inia 66), was obtained from a nitrogen fertilizer field trial grown in Mexico. A high positive correlation was obtained between seed protein content and seedling dry weight after 3 weeks growth (r = +0.92^**). The seedling dry weight was positively related to the protein content of the aleurone layer and endosperm, but not to the embryo. Small, 35 milligrams, high protein seeds (4.7 milligrams protein per seed) produced larger seedlings than large, 45 milligrams, low protein seeds (4.3 milligram protein per seed). There was no difference in the weight or protein content of embryos from low and high protein seeds and their growth was similar. Composite seeds of the two protein levels were produced by transferring embryos from one endosperm type to the other. After 4 weeks, there was no difference between the different embryo types grown on the same endosperm type. High protein endosperm produced more vigorous seedlings regardless of the embryo type grown on it, indicating that the factor(s) responsible for the greater growth of high protein seed is in the endosperm.     

Molecular characterization and physico-chemical analysis of a new giant embryo mutant allele (ge t) in rice (Oryza sativa L.)

The rice embryo is rich in lipid and protein bodies, bioactive chemicals such as dietary fiber, phytic acids, vitamin B and E, and gamma aminobutyric acid (GABA) than the endosperm. In this paper, we report a new giant embryo mutant,ge ^t, induced from somaclonal variation derived by anther culture in rice. Sequence analysis of Cytochrome P450 of the get mutant revealed thatge ^t is a new allele of theGE gene with a single point mutation with substitution of amino acid, W³⁹⁵ to L³⁹⁵. The weight of thege ^t mutant embryo was 3.7 times higher than normal embryo. Tocopherol and mineral content were also higher than the previously reported giant embryo rice variety, Keunnun. These results indicated that this new giant embryo rice (ge ^t) offers a promising source of genetic material in improving nutritional quality of rice especially tocopherol, essential minerals, and GABA.     

Degradation of the endosperm cell walls of Lactuca sativa L., cv. Grand Rapids

The timing of mobilisation of lipid, sucrose, raffinose and phytate in lettuce seeds (achenes) (cv. Grand Rapids) has been examined. These reserves (33%, 1.5%, 0.7%, 1.4% of achene dry weight, respectively) are stored mostly in the cotyledons. Except for a slight degradation of raffinose and increase in sucrose, there is no detectable reserve mobilisation during germination. The endosperm (8% of seed dry weight), which has thick, mannan-containing cell walls (carbohydrate, 3,4% of seed dry weight), is completely degraded within about 15h following germination. Mannanase activity increases about 100-fold during the same period and arises in all regions of the endosperm. Also during this period sucrose and raffinose are degraded and fructose and glucose accumulate in the embryo. The endosperm hydrolysis products are taken up by the embryo, and are probably used as an additional reserve to support early seedling growth. However, endosperm cell-wall carbohydrates, such as mannose, are not found as free sugars. Lipid and phytate are degraded in a later, second phase of mobilisation. Low levels of sucrose are present in the embryo, mostly in the cotyledons, and large amounts of fractose and glucose (14% of seedling dry weight at 3 days after sowing) accumulate in the hypocotyl and radicle. It is suggested that sucrose, produced in the cotyledons by gluco-neogenesis, is translocated to the axis and converted there to fructose and glucose.     

Subcellular distribution of gluconeogenetic enzymes in germinating castor bean endosperm

The intracellular distribution of enzymes capable of catalyzing the reactions from oxaloacetate to sucrose in germinating castor bean endosperm has been studied by sucrose density gradient centrifugation. One set of glycolytic enzyme activities was detected in the plastids and another in the cytosol. The percentages of their activities in the plastids were less than 10% of total activities except for aldolase and fructose diphosphatase. The activities of several of the enzymes present in the plastids seem to be too low to account for the in vivo rate of gluconeogenesis whereas those in the cytosol are quite adequate. Furthermore, phosphoenolypyruvate carboxykinase, sucrose phosphate synthetase, and sucrose synthetase, which catalyze the first and final steps in the conversion of oxaloacetate to sucrose, were found only in the cytosol. It is deduced that in germinating castor bean endosperm the complete conversion of oxaloacetate to sucrose and CO₂ occurs in the cytosol. The plastids contain some enzymes of the pentose phosphate pathway, pyruvate dehydrogenase and fatty acid synthetase in addition to the set of glycolytic enzymes. This suggests that the role of the plastid in the endosperm of germinating castor bean is the production of fatty acids from sugar phosphates, as it is known to be in the endosperm during seed development.     

Effect of sucrose accumulation on zein synthesis in maize starch-deficient mutants

Starch-deficient maize (Zea mays) mutants, brittle-2 (bt2), brittle-1 (bt), and shrunken-2 (sh2), which accumulated large quantities of sucrose, had less than normal amounts of zein (the major storage protein) in the endosperm. Reduction of zein synthesis in the starch-deficient mutants was negatively correlated with the accumulation of sucrose and low osmotic potential in the developing endosperms. When radioactive amino acids were injected into the shank below ears that segregated for the starch-deficient mutant and normal kernels at 28 days post-pollination, mutant kernels absorbed only ca 22–36% of the labelled amino acids found in their normal controls. Thus, a low osmotic potential in the mutant endosperm may favour water movement but reduce solute movement. The inability of amino acids to move into the mutant endosperms, therefore, in part explains the reduction of zein accumulation in starch-deficient mutant endosperms.     

Growth and composition of maize kernels cultured in vitro with varying supplies of carbon and nitrogen

This study employed in vitro seed culture to determine how C and N supply influence the growth (i.e. starch accumulation) and protein composition of maize (Zea mays L.) endosperm. Immature kernels were grown to maturity on liquid medium containing various concentrations of C (sucrose at 234 millimolar [low] and 468 millimolar [high]) and N (amino acid mixture ranging in N from 0 to 144 millimolar). Low C supply limited starch, but not N, accumulation in the endosperm. With high C, endosperm starch and protein content increased concomitantly as N supply increased from 0 to 13.4 millimolar. Endosperm growth was unaffected by additional N until concentrations exceeding approximately 72 millimolar reduced starch accumulation. A similar inhibition of starch deposition occurred with lower N concentrations when kernels were grown with low C. Endosperm total N content reached a point of saturation with approximately 36 millimolar N in the medium, regardless of C supply. Zein synthesis in the endosperm responded positively across all N levels, while glutelin content remained static and albumin/globulin proteins were reduced in amount when N supply was greater than 36 millimolar. A reciprocal, inverse relationship was observed in mature endosperm tissue between the concentrations of free amino acids and soluble sugars. Our data suggest that under N stress starch and protein accumulation in the endosperm are interdependent, at least in appearance, but are independent otherwise.     

Dynamic of reserve compounds of mesocarp and seeds of macaw palm (<Emphasis Type="Italic">Acrocomia aculeata)</Emphasis> submitted to different storage conditions

Key message

Storage changed carbohydrate and protein levels, which can be related to embryo viability. The fatty acid profile was constant, and the embryo composition was similar to the mesocarp, not the endosperm.

Abstract

Macaw palm fruits have a diverse biochemical constitution, and there is significant commercial interest in this species among food, pharmaceutical, cosmetics, and bioenergy industries. We evaluated changes in the reserve compounds of macaw palm mesocarp and seeds from fruits stored for 1 year under three different conditions. Protein and carbohydrate levels were highest in the embryo than in the endosperm. Fatty acid profiles were very similar over time under all storage conditions and in each structure evaluated, with the embryo composition being very similar to the mesocarp. Macaw palm oil remained well preserved under all storage conditions tested, but seed reserves and seed viability are best maintained at room temperatures. The endosperm contained higher levels of saturated fatty acids than either the embryo or mesocarp, making seeds more resistant to oxidative deterioration than the mesocarp. The results showed that the composition of the mesocarp oil promises the production of high-quality biodiesel from this structure, and changes in carbohydrate and protein levels show that laboratory conditions are the most efficient for maintaining seed quality during storage.