The Metabolic Role of the Legume Endosperm · The Metabolic Role of the Legume Endosperm: A...

The Metabolic Role of the Legume Endosperm:A Noninvasive Imaging Study1[W][OA]

Gerd Melkus2, Hardy Rolletschek2, Ruslana Radchuk, Johannes Fuchs, Twan Rutten, Ulrich Wobus,Thomas Altmann, Peter Jakob, and Ljudmilla Borisjuk*

University of Wurzburg, Institute of Experimental Physics 5, 97074 Wuerzburg, Germany (G.M., J.F., P.J.); andLeibniz-Institut fur Pflanzengenetik und Kulturpflanzenforschung, 06466 Gatersleben, Germany (H.R., R.R.,T.R., U.W., T.A., L.B.)

Although essential for normal seed development in the legumes, the metabolic role of the endosperm remains uncertain. Wedesigned noninvasive nuclear magnetic resonance tools for the in vivo study of key metabolites in the transient liquidendosperm of intact pea (Pisum sativum) seeds. The steady-state levels of sucrose, glutamine, and alanine could be monitoredand their distribution within the embryo sac visualized. Seed structure was digitalized as a three-dimensional model,providing volume information for distinct seed organs. The nuclear magnetic resonance method, combined with lasermicrodissection, isotope labeling, in situ hybridization, and electron microscopy, was used to contrast the wild-typeendosperm with that of a mutant in which embryo growth is retarded. Expression of sequences encoding amino acid andsucrose transporters was up-regulated earlier in the endosperm than in the embryo, and this activity led to the accumulation ofsoluble metabolites in the endosperm vacuole. The endosperm provides a temporary source of nutrition, permits space forembryo growth, and acts as a buffer between the maternal organism and its offspring. The concentration of sucrose in theendosperm vacuole is developmentally controlled, while the total amount accumulated depends on the growth of the embryo.The endosperm concentration of glutamine is a limiting factor for protein storage. The properties of the endosperm ensure thatthe young embryo develops within a homeostatic environment, necessary to sustain embryogenesis. We argue for a degree ofmetabolite-mediated control exerted by the endosperm on the growth of, and assimilate storage by, the embryo.

The seed represents the link between the genera-tions in higher plants. The seed coat, derived frommaternal tissue, serves to both protect the filial gener-ation and act as a conduit for the supply of nutrients(Murray, 1987). The diploid embryo and triploid en-dosperm both originate from the double fertilizationevent (Bewley and Black, 1994), but the developmentalfates of these two organs differ greatly from oneanother. The embryo progresses through a fixed pat-tern of cellular divisions to form a series of specializedorgans, whereas the endosperm, which initially growscoenocytically, either becomes a major storage organ(monocotyledonous species) or degenerates (dicotyle-donous species) as the seed matures (Olsen, 2004;Sabelli and Larkins, 2009). The monocotyledonousendosperm serves as a crucial provider of nutritionduring germination, while the role of the transient

dicotyledonous endosperm has remained a puzzledespite more than a century of research.

The importance of the endosperm has become evi-dent thanks to the elucidation of the molecular mech-anisms underlying the control of seed size (for review,see Chaudhury et al., 2001; Berger et al., 2006). A seriesof genetic and epigenetic interactions in the endospermis now known to play a crucial regulatory role overembryogenesis (Gehring et al., 2004; Nowack et al.,2007). The triploid endosperm appears to be a battle-ground over how resources are allocated to the embryo(Haig and Westoby, 2006). Given the higher dosage ofmaternal over paternal chromosomes, the mothermay be able to exert greater control over nutrient allo-cation to the progeny and, in so doing, maximize herown fitness. The triploid state of endosperm appears tobe advantageous for the transfer of nutrients from thematernal to the filial generation (Stewart-Cox et al., 2004;Sundaresan, 2005). Many genes/transcription factorsactive in the endosperm have been identified as havingan effect on seed size (Ohto et al., 2005; Kondou et al.,2008), whereas the embryo contributes comparativelylittle to this trait (Garcia et al., 2005; Ingouff et al., 2006).The growth of the seed relies on a degree of coordinationexercised by the endosperm, but how this is achieved atthe metabolic level remains unclear.

The small seed size of the model plants Arabidopsis(Arabidopsis thaliana) and Medicago truncatula presentsa technical challenge for the analysis of endospermcomposition. The application of laser microdissection

1 This work was supported by the Federal Ministry of Educationand Research.

2 These authors contributed equally to the article.* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Ljudmilla Borisjuk ([email protected]).

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a sub-

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(Schneider and Holscher, 2007) and/or noninvasivefluorescence resonance energy transfer nanosensors(Looger et al., 2005) can address some of these diffi-culties but does not help in the context of metabolicanalysis, for which large-seeded species represent amore tractable subject (Zhang et al., 2007). Seed de-velopment in the agriculturally important species pea(Pisum sativum), soybean (Glycine max), broad bean(Vicia faba), and rapeseed (Brassica napus) has beenextensively studied; thus, a preexisting body of knowl-edge surrounds both the spatial and temporal distri-bution patterns of metabolites in the embryo (forreview, see Borisjuk et al., 2003) and nutrient loadinginto the seed (for review, see Patrick and Offler, 2001).However, the composition of the liquid endosperm andseed apoplast has only been touched upon (Giffordand Thorne, 1985; Borisjuk et al., 2002; Hill et al., 2003).This lack of experimental data largely reflects the diffi-culty of noninvasive sampling, without which the com-partmentalization of metabolites and the biochemistrywithin the liquid endosperm cannot be determined.Current NMR technology now allows for the noninva-sive study of metabolites in cell cultures, animals, andplants (Bourgeois et al., 1991; Metzler et al., 1995; Tseet al., 1996; Kockenberger, 2001; Neuberger et al., 2008).

Here, we sought to develop an analytical tool for thenoninvasive imaging, quantification, and monitoringof metabolites in the living endosperm and to use thisinformation to investigate the metabolic role of theendosperm in the legume seed. We have combinednoninvasive NMR methods with more conventionalhistological and biochemical analyses to characterizethe pea endosperm in both the wild type and a mutantin which the epidermal cell identity in the embryo hasbeen lost (Borisjuk et al., 2002). The development of agiant endosperm in this mutant facilitates the elucida-tion of maternal-filial interactions and the mode ofsolute transport within the seed. The combination ofapproaches allowed a literally “new view” of thetransient endosperm, its interrelation in vivo, and itsfunction in legume seed.

RESULTS

Endosperm as an Immediate Environment of Embryoin Vivo

During its early development, the pea endospermforms a motile, proliferating tissue lining the embryosac cavity, later enveloping the embryo and suspensor(data not shown; Marinos, 1970). Endosperm becomesenlarged to occupymost of the embryo sac and forms alarge vacuole in the chalazal region (Fig. 1A). Since thevacuole is completely enveloped by the endospermcytoplasm, it belongs, spatially at least, to the endo-sperm and therefore can be considered as an “endo-sperm vacuole.” Cellularization of the endospermcytoplasm occurs during the mid stage of develop-ment, at which time the endosperm vacuole becomessurrounded by a cell wall (Fig. 1, B and C), so that its

status alters to become an apoplastic compartmentwithin the endosperm. An NMR scan of a livingendosperm vacuole (Fig. 1, D–I; Supplemental MoviesS1–S3) shows a strongly labeled body (liquid-filledspace) that embeds both the embryo and the suspensorthroughout development. The layers of the endospermcytoplasm could not be spatially resolved and appearto remain closely attached to the embryo sac wall andembryo surface. No air-filled spaces or other struc-tures within the endosperm vacuole were identifiable.Early in development, the embryo floats within theendosperm vacuole (Fig. 1, D–I), thereby insulating itfrommechanical pressure imposed by the seed coat. Thesuspensor binds the embryo to the embryo sac bound-ary wall, and its expansion pushes the embryo awayfrom themicropyle (Fig. 1, D andG) and toward themid(Fig. 1, E and H) or lower (Fig. 1, F and I) part of theembryo sac. The direct connection between the embryoand the maternal tissues is disturbed as the suspensorloses its integrity. The conventional assumption that theembryo is anchored to the boundary wall of the embryosac (Marinos, 1970), therefore, needs to be reassessed.

The NMR data were used to reconstruct a digitalmodel of individual seeds (Fig. 2, A–C; SupplementalMovie S4), which permitted a three-dimensional visu-alization of seed anatomy and, in particular, allowedfor the measurement of the volume of various seedorgans and the detection of any changes in theirrelative size over time (Fig. 2, D–F). During a periodover which the seed volume of the wild-type peaexpanded 4-fold, the suspensor disappeared while thesize of the embryo increased. The size ratio betweenthe endosperm and the seed coat did not vary. Thegrowth of seed is coupled with the major increase inseed coat volume, in concert with the rapid growth ofthe endosperm. In the E2748 mutant (SupplementalFig. S1, A and B), the early stages of embryogenesis aresimilar to those occurring in the wild type, but fromthe mid cotyledon stage onward, embryo growth stopsand a callus-like body is formed (Borisjuk et al., 2002).

The Growth of the Seed Is Primarily Associated with theInitial Growth of the Endosperm

The embryo-endosperm ratio was assessed in seedsdeveloping on heterozygotic mutant plants (Mm).The phenotype of the progeny of these plants waseither wild type (Mm and MM) or mutant (mm; Sup-plemental Fig. S1C). Intact seeds appear identical, butmagnetic resonance imaging (MRI) was able to differ-entiate between the mutant and the wild type, thusavoiding the need for dissection (Fig. 3, A, B, E, and F).The seed size and endosperm volume of the two typesare similar up to a seed freshweight of 30 to 50mg (datanot shown). Thereafter, morphological differences be-come apparent in the embryo sac, although not in theseed coat. By approximately 150 to 200 mg seed freshweight, wild-type embryos have reached the samemass (volume) as their endosperm (“equilibrationpoint”) and continue to grow at the expense of the

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endosperm (Fig. 3D). The wild-type seed grows to afresh weight of 450 mg, at which point the embryoalmost completely fills the seed (Fig. 3C). By this latterstage, all of the major developmental events have beenachieved (stage 24, as identified by Marinos, 1970) andfurther increases in seed size are minimal. The mutantembryo’s growth is retarded, although the volume ofthe liquid endosperm continues to increase (Fig. 3H).By the time the mutant seed fresh weight has reachedapproximately 150 to 200 mg, the endosperm hasbecome 3- to 5-fold larger than the embryo; at a freshweight of 300 mg, the endosperm ceases its growth andbegins to dehydrate to form a shrunken seed (Fig. 3G).Thus, the growth of the seed is primarily associatedwith the early growth of the endosperm rather thanwith any later growth of the embryo.

Retardation in Embryo Growth in the Mutant Seed DoesNot Induce Starch/Protein Storage in the Endosperm

In both the wild-type and mutant seed, histologicaland immunological analysis showed that both thedeposition of starch granules and the accumulation ofstorage protein are restricted to the embryo (Supple-mental Fig. S2, A–D). Transmission electron micros-

copy (Supplemental Fig. S2, E and F) confirmed thatonly a small number of lipid droplets are accumulatedin the endosperm and also identified the formation ofmultiple endoplasmic reticulum and Golgi structures,each surrounded by mitochondria. Together, these or-gans represent the subcellular machinery utilized by abiosynthetically active cytoplasm. Even when embryogrowth and storage activity are retarded, the endospermretains its characteristics as a transient organ and doesnot coopt any of the storage functions of the embryo.

Experimental Design for the in Vivo Imaging

of Metabolites

Noninvasive 1H-NMR localized spectroscopy wasused to analyze the in vivo composition of the endo-sperm vacuole in both wild-type and mutant seeds.1H-NMR metabolites were detected in the endospermby voxel-selective point resolved spectroscopy (PRESS;Bottomley, 1984). Suc, Ala, Gln, lactate, and Val allappear as well-resolved peaks (Fig. 4A). Localized two-dimensional correlation spectroscopy (L-COSY; Fig. 4, Band C) was used to clarify the overlapping spectralrange (3.4–4.4 ppm). The L-COSY pulse sequence isgiven in Supplemental Fig. S3. 1H-NMR chemical shift

Figure 1. Anatomy of seeds as vi-sualized by conventional histologyand noninvasive MRI. Images arerepresentative for approximately30 to 50 mg fresh weight. A, His-tostaining of the cross sectionthough the chalazal region of aseed (approximately 30 mg freshweight) shows the embryo, endo-sperm, and endosperm vacuolesurrounded by endosperm, embryosac walls, and seed coat. Due totoluidine blue staining, seed tissuesexcept the endosperm vacuole areblue stained. B, Fragment of a sec-tion though the micropylar regionof a seed (approximately 50 mgfresh weight) demonstrates cellu-larized endosperm, which en-velops the embryo and bordersthe embryo sac wall (arrows). To-luidine blue staining is shown. C,The same as in B but within thechalazal region of a seed. D to I,Fragments of three-dimensionalimaging using MRI (SupplementalMovie S1) showing internal struc-tures of a seed. The noninvasiveviews correspond to the longitudi-nal section (D–F) and cross section(G–I). e, Embryo; en, endosperm;ev, endosperm vacuole; s, suspen-sor; sc, seed coat.

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imaging (CSI) with high spatial resolution (1003 1003100 mm3) was applied on intact seeds at an earlystage of development to image metabolite distribu-tion. Structural FLASH (for fast low angle shot)multislice images were acquired at the end of the CSIprotocol in order to topographically relate the spec-troscopic data with the corresponding tissue struc-tures at each pixel of the image.

The Spatial Distribution of Suc Differs from That ofAmino Acids

Suc, Gln, and Ala are the major nutrients supplied tothe zygote by the maternal plant. The distribution ofSuc within the intact embryo sac at the early cotyledonstage showed that the highest concentrations are pres-ent in the central (adaxial) region of the cotyledon,decreasing toward its periphery (Fig. 5, A and B). Sucdistribution within the endosperm vacuole is ratheruniform. The lowest Suc levels coincide topographi-cally with the suspensor. The distribution of Ala isquite distinct from that of Suc. The highest level ispresent in the endosperm and the suspensor, with verylittle accumulating in the cotyledon (Fig. 5, A and C).The distribution of Gln is identical to that of Ala (datanot shown). Additionally, NMR spectra were obtainedfrom three distinct regions of the liquid endosperm:one surrounding the embryo, one surrounding themicropyle, and the last surrounding the chalazal re-gion (Supplemental Fig. S4). The acquired spectra didnot differ significantly from one another, in accordancewith the imaging data set. Thus, the liquid endospermappears to be rather homogeneous, in contrast to thecontent of the cellularized embryo.

NMR-Based Tools to Monitor Metabolites in theIntact Endosperm

The spectral parameters of the intact endospermat the early and late stages of development differmarkedly from one another (Supplemental Fig. S5),indicating that compositional changes do occur. Asthe purpose of these experiments was to quantify indi-vidual metabolite levels during development, the NMRsignal amplitudes, acquired via the low-resolution CSImethod, were converted into metabolite concentrationsusing a phantom replacement method (Soher et al.,1996). An external standard of known Suc, Ala, and Glnconcentrations was measured using the in vivo protocoland used as a reference to correct the in vivo signal (fordetails, see “Materials and Methods”). For standardiza-tion purposes, the endosperm samples were isolatedand analyzed biochemically immediately following theMRI experiment. The NMR in vivo Suc, Ala, and Glusignals were plotted against their concentrations ob-tained by the corresponding biochemical assay (Supple-mental Fig. S6), and the resulting correlations were allhigh (Suc, r2 = 0.99; Gln, r2 = 0.93; Ala, r2 = 0.63).

The Accumulation of Suc and Amino Acids in theLiquid Endosperm Follows Distinct Temporal Patternsduring Development

The in vivo concentration of metabolites in theliquid endosperm of wild-type and mutant seedswas measured over the course of development (Fig.6). The concentration of Suc increases steadily toapproximately 250 mM until seed fresh weight reaches150 to 170 mg and remains stable thereafter (Fig. 6A).

Figure 2. Digital models of three-dimensional anatomy of a developingpea seed at different developmentalstages based on the in vivo NMR dataset. Seed coat, endosperm, suspensor,and embryo were identified and colorcoded. A to C, Images represent seedsof 30, 73, and 120 mg fresh weight.Different seed tissues are color coded.D to F, Volumes of different seed tis-sues calculated using AMIRA software:seed coat (green), endosperm (blue),embryo (red), suspensor (yellow). e,Embryo; en, endosperm; s, suspensor;sc, seed coat.

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The dynamics of Suc accumulation are similar in thewild-type and mutant seeds, but there are large dif-ferences in the accumulation of amino acids. Duringearly development, both Gln and Ala levels increase(Fig. 6, B and C). However, from a seed fresh weight of80 to 120 mg, both Gln and Ala levels decreasesignificantly in the wild type but continue to rise inthe mutant. By the time seed fresh weight has reached

250 mg, the Gln and Ala concentrations in the wild-type endosperms are severalfold lower than in themutant. Thus, Suc and amino acid levels in the endo-sperm appear to be under distinct control.

Sequences Encoding the Amino Acid and Suc

Transporters Are Differentially Expressed in theMaternal and Filial Tissues of Individual Seeds

The accumulation of high concentrations of aminoacids and Suc in the endosperm vacuole suggested thatthe uptake of assimilates in the endosperm is active.Amino acid permeases (such as AAP1 and AAP2;Tegeder et al., 2000) and Suc transporter/facilitator(SUF1 [Zhou et al., 2009] and SUT1 [Tegeder et al.,1999]), both of which are involved in assimilate trans-port in the legume seed, have been functionally char-acterized. However, these have all been localized inonly the embryo and/or seed coat of pea, while theirpresence in the endosperm has not to date been inves-tigated. To determine this, therefore, maternal seed coatand filial tissues (endosperm, embryo) were isolatedusing laser-assisted microdissection from seeds of ap-proximately 35 mg fresh weight (Fig. 7, A–E), and theresulting RNA populations were analyzed by reversetranscription (RT)-PCR. The outcome of these experi-ments on individual deeds showed that PsAAP1,PsSUF1, and PsSUT1 transcripts are present in endo-sperm but not in embryo (Fig. 7F). PsAAP2 transcriptswere observed at the limit of detection in the seed coat,endosperm, and embryo. Thus, the genes directing thetransport of amino acids and Suc are up-regulated inendosperm tissue earlier than in the embryo.

The Spatial Expression Pattern of Sut1 in the EndospermIs Consistent with the Delivery of Suc from theSeed Coat

A Sut1-specific sequence was used as a probe for thein situ localization of Sut1 expression in the endo-sperm during the early stages of seed development.This showed that transcripts are more abundant inthe endosperm than in the embryo (Fig. 8, A–D), inaccordance with the RT-PCR outcome (Fig. 7F). Themajor zone of expression lies at the peripheral surfaceof the endosperm cytoplasm, with only a modest levelof expression occurring in the endosperm cytoplasmattached to the embryo. Thus, the external and internalendosperm membranes appear to differ from oneanother functionally with respect to Suc transport(arrows in Fig. 8A). The localization of Sut1 expressionto the embryo sac-endosperm interface implicates thisregion as being important for the transport of Suc fromthe maternal apoplast to the endosperm vacuole. Torelate Sut1 expression to Suc levels in the endosperm,total Suc amounts were calculated from the productof its concentration and the vacuole volume (Fig. 8E).The up-regulation of Sut1 in the endosperm coincidestemporally with the onset of Suc accumulation inthe vacuole. Thus, the endosperm appears capable of

Figure 3. Developmental parameters of E2748 mutant (mm) and wild-type (Mm) phenotype seeds. A, Seed of wild-type phenotype bynoninvasive NMR. B, The same seed but dissected by hand showingendosperm vacuole (ev) and embryo (e). C, Section through matureseed where embryo sac is occupied by embryo. D, Fresh weight (fw)accumulation of embryos (squares) and volumes of the endospermvacuole (circles) in wild-type seed. Data points represent single mea-surements. E, Seed of mutant phenotype by noninvasive NMR. F, Thesame seed but dissected by hand showing liquid endosperm (ev) andembryo (e). G, Mature seed with characteristic shrunken phenotype. H,Fresh weight accumulation of embryos (squares) and endospermvacuole volumes (circles) in mutant seed. Data points represent singlemeasurements. The orange hatched lines in D and H indicate the stagesof seeds shown in A/B and E/F, respectively.





acquiring Suc transport activity and begins to accu-mulate Suc before the embryo does. From the midcotyledon stage onward, the expression shifts to theembryo (Tegeder et al., 1999; Borisjuk et al., 2002).

Endosperm Metabolite Levels Respond to the Onset of

Storage Activity in the Embryo

An analysis of the temporal pattern of starch andprotein accumulation in the embryo enabled the clar-ification of the relationship between assimilate leveland storage activity. In the wild type, starch depositionbegins at a low rate once the seed fresh weight hadreached 120mg but rises as seed freshweight increasesfrom 150 to 200 mg (Fig. 9A). The Suc concentration inthe endosperm vacuole does not respond to the initi-ation of starch storage in the embryo, but the amountof Suc in the wild-type endosperm declines markedlyfrom this time onward (Fig. 8E). In the mutant endo-sperm, Suc content remains high (Fig. 8E), correspond-ing to a much lesser amount of starch being stored inembryo (Borisjuk et al., 2002). However, the Suc con-centration was similar to that in wild-type endosperm(Fig. 9A). Thus, the Suc reserves in the endospermappear to be accessed by the growing embryo, buthomeostatic mechanisms then come into play to con-trol/balance its concentration.

Protein storage was initiated in the wild-type seedonce its fresh weight reached 100 to 150 mg (Fig. 9B),achieving a peak rate at 200 to 250 mg. The increase inprotein storage activity coincides with a fall in the levelof Gln (and Ala) in the endosperm vacuole. In mutantseeds, the accumulation of protein per embryo is muchreduced (Borisjuk et al., 2002). Correspondingly, theendosperm vacuole level of Gln in the mutant seedremains high over a sustained period during devel-opment (Fig. 9B).

The developmental decline in the endosperm Gln(Ala) concentration coincides temporally with risingdemands of the growing embryo and therefore mayserve to limit the uptake of amino acids by the embryo, atthe same time depressing the rate of storage proteindeposition. In an in vitro experiment designed to deter-mine the limiting endosperm level of Gln, intact wild-type embryos were incubated in the presence of variousconcentrations of 15N-labeled Gln (while the concentra-tions of Ala, Suc, and inorganic compounds were main-tained at their in vivo levels). Increasing the availablelevel of Gln from 10 to 60 mM enhanced the 15N signal inthe protein fraction (Fig. 9C), but further increases hadno effect. Because the in vivo endosperm Gln concen-tration declines to below 60mMduring development, theindication is that protein storage in the embryo becomeslimiting from a seed fresh weight of 100 to 150 mg.

Metabolite Levels in the Endosperm in Response tothe Environment

A series of experiments was designed to explorewhether the level of nutrients present in the endo-

Figure 4. Localized NMR spectra from the endosperm of wild-typeseeds (approximately 260 mg fresh weight). A, PRESS spectrum.Resonances from the metabolites Suc, Ala, Gln, lactate (Lac), and Valare visible. B, L-COSY spectrum. The cross-peaks in the spectrum(indicated by the metabolite name labeling) confirm the assignedmetabolites in A. The white square indicates the enlarged spectrum,which is presented in C. C, Enlarged spectrum from B. The cross-peaksdetected in the range 3.2 to 4.2 ppm result from Suc. No othermetabolites were detected in this spectral area.

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sperm is sensitive to the external environment. Theeffect of nutrient starvation was tested by removingintact seeds (150 mg fresh weight) from the pod andusing NMR to monitor sugar and free amino acidcontents in the endosperm vacuole. Over a 4-h period,metabolite levels in the endosperm remained unaf-fected by the disconnection of nutrient supply (Sup-plemental Fig. S7). Next, the nutrient level present in a100-mg seed fresh weight endosperm in the middle ofthe day was compared with that present in the middleof the night. The Suc concentration varied between 170and 194 mM, and that of Gln between 64 and 75 mM,but critically, the level appears to be independent oftime of day. Finally, detached seeds were exposedfor 2 h to either 100% oxygen or 100% nitrogen toexamine the effect of exogenous oxygen concentrationon the endosperm nutrient level. This treatment has

been shown to have a large effect on nutrient uptakeand respiratory and storage metabolism of seed(Rolletschek et al., 2005b). However, neither hyperoxianor anoxia materially altered the levels of Suc, Gln, orAla (data not shown). Thus, the liquid endosperm thatsurrounds the developing embryo represents a me-dium where the nutrient level remains balanced evenin the face of major changes in the external environ-ment.

DISCUSSION

Recent research has identified the endosperm as anintegrator of genetic programs controlling seed devel-opment (Berger et al., 2006) and has suggested that itsmetabolic role needs to be reinterpreted. Here, we

Figure 5. Noninvasive mapping of metabolites inembryo sac of pea seed (approximately 50 mg freshweight). A, Visualization of embryo sac by MRIshows positioning of seed coat, embryo, endosperm,and suspensor. Small boxes in pink show the regionchosen for spectroscopic analysis (see right panels).B, Visualization of Suc distribution within the embryosac. The bar represents the color code for specificsignal intensity. C, Visualization of Ala distributionwithin the embryo sac. The bar represents the colorcode for signal intensity. Graphs at right show spec-tral characteristics of different regions of embryo sac:embryo, endosperm, and suspensor. e, Embryo; ev,endosperm vacuole; m, micropyle; s, suspensor; sc,seed coat.





have applied NMR to investigate the metabolism ofthe liquid endosperm in vivo within the developingpea seed. Our study argues for a metabolic role of theendosperm in legume seeds.

Advantages and Limitations of the Noninvasive

Measurement of Endosperm Metabolite Levels

Physical access to the liquid endosperm is muchmore problematical than to the embryo (Borisjuk et al.,2003; Hill et al., 2003; Morley-Smith et al., 2008). Arecognized disadvantage of invasive sampling is itsinduction of a wounding response, quite apart fromthe inevitable disruption of the spatial distribution ofmetabolites. The resulting artifactual concentrations ofmetabolites can be disturbed by 1 order of magnitude(for review, see Gifford and Thorne, 1985), and as aresult, little effort has been made to systematicallystudy metabolite levels in the liquid endosperm. MRItechnology, however, provides a noninvasive means ofmeasuring metabolite concentrations (Bourgeois et al.,1991; Soher et al., 1996; Vanhamme et al.,1997; Tkacet al., 1999; Pohmann and von Kienlin 2001; De Graaf,2007).

The use of NMR to track some of the major seedmetabolites has a number of advantages. First, theseed remains undamaged by the process of samplepreparation, so wound responses are avoided; second,the use of a CSI method with a direct free inductiondecay acquisition and a long repetition time allows formetabolite quantification with a minimal requirementfor postprocessing correction; and finally, the acquisi-tion and processing procedure is simple and robust,because only one pulse and a phase-encoding gradientare needed for signal encoding. Some plant tissues,however, are refractory to MRI (Ratcliffe and Shachar-Hill, 2001; Kockenberger et al., 2004), and the methodappears particularly unsuited for the analysis of low-concentration metabolites. Achieving good levels ofsensitivity in small seeds will need modification ofcurrently available coils (Neuberger and Webb, 2009).

In Vivo Imaging Reveals an Absence of MetaboliteGradients in the Liquid Endosperm But Their Presencein the Embryo

The NMR analysis captured the in vivo state of theseed at a distinct phase of its development. In partic-ular, it was possible to identify the presence of a Sucgradient within the embryo, falling from a high levelin the central (adaxial) part of the cotyledon to a lowlevel at the periphery. This gradient correspondedspatially with the differentiation pattern of the embryo(Hauxwell et al., 1990; Borisjuk et al., 2002, 2003).Differentiation of the endosperm has been describedat both the cytological (Boisnard-Lorig et al., 2001) andgene expression (Fig. 8; Tanaka et al., 2001; Bate et al.,2004) levels. However, the endosperm vacuole appearsto be rather homogeneous with respect to Suc, Ala,and Gln (Fig. 5; Supplemental Fig. S4), suggesting thatit represents a large homogenous compartment. Incontrast to what pertains in the embryo tissue, equil-ibration within the endosperm vacuole can take placerapidly, thus allowing for an efficient transfer of nu-trients from the source (seed coat) to the sink (embryo).

Figure 6. Steady-state in vivo levels of metabolites in endospermvacuoles of developing seeds measured by noninvasive NMR. A, Suc.B, Ala. C, Gln. WT, Wild type.

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This scenario may well be advantageous in the contextof the seed’s energy economy (Borisjuk and Rolletschek,2009) and may favor metabolite exchange between twogenerations (Lersten, 2004).The suspensor, which connects the embryo to the

mother plant (Fig. 2; Supplemental Movies S1 and S2),contains similar Ala/Gln levels as the endosperm, butits level of Suc is substantially lower (Fig. 5B). Thus,the embryo is exposed to a particular Suc level at itsattachment region to the suspensor. Since Suc can actas a signal molecule (Chiou and Bush, 1998), this maylocally affect cell differentiation (Rolland et al., 2006) inconcert with the suspensor’s function in embryo pat-terning, as postulated from in vitro studies (Supenaet al., 2008).

The Role of Suc in Determining the HomeostaticProperties of the Endosperm

Suc uptake is responsible for approximately 70% ofthe dry matter in the mature pea seed (Patrick andOffler, 2001). Its rate is determined by the activity ofSuc transporters (Tegeder et al., 2000; Rosche et al.,2002), which in turn is largely controlled by theembryo’s carbohydrate demand (Zhou et al., 2009).The up-regulation of Sut1 in the embryo, which isprimarily localized within the outer regions of thecotyledons, is coupled with an increase in assimilate

sink strength (Borisjuk et al., 2002). Our data havedemonstrated that Sut1 is also expressed in theendosperm, where its up-regulation anticipates itsexpression in the embryo, and that its expression islargely confined to the region adjacent to the seedcoat. The endosperm’s surface area appears ratherlarger than that of its partner embryo (Fig. 2A). Sut1expression in the endosperm is followed by a rapidaccumulation of Suc in the endosperm vacuole.Even though similar events take place in the embryo,the endosperm grows much more rapidly than theembryo does. Thus, it is reasonable to suppose thatthe endosperm is predetermined as a primary sinkfor Suc and follows a similar strategy in terms ofSuc uptake as does the embryo. A defective mutationin the Arabidopsis endosperm-specific Suc trans-porter AtSUC5, which promotes the rate of Suctransport into the endosperm, has been associatedwith a delay in embryo development (Baud et al.,2005). We have shown here that the amount of Suc inthe endosperm is inversely proportional to the rateof embryo growth and storage: starting from approx-imately 150 to 200 mg seed fresh weight (“equilib-rium point”), the total amount of Suc in endospermdecreases (Fig. 8E), whereas starch accumulation ratein embryo increases (Fig. 9A). This indicates the roleof the endosperm as a carbohydrate source for theembryo.

Figure 7. Tissue-specific analysis oftransport-related genes using laser-assisted microdissection and RT-PCRof pea seed (approximately 35mg freshweight). A to C, Dissection procedurefor embryo: before dissection (A), afterdissection (B), and dissected embryofragment (C). The red line in A indi-cates tissue chosen for dissection. Dand E, Dissection of seed coat (D) andendosperm (E). The green lines indi-cate tissue chosen for dissection. F,Results of RT-PCR analysis of PsAAP1,PsAAP2,PsSUF1, andPsSUT1 genes ex-pressed in endosperm, embryo proper,and seed coat. Amount of cDNA usedper sample for PCR as a template wasapproximately 20 ng (see “Materialsand Methods”). Actin mRNA abun-dance reflects approximate, but notexact, amount of cDNA template usedfor RT-PCR. e, Embryo; en, endosperm;sc, seed coat.





In contrast to the total amount of Suc present in theendosperm vacuole, its steady-state level is not af-fected by the growth of the embryo (Fig. 9A). Thetemporal gradient of Suc suggests that its steady statein the endosperm vacuole is developmentally fixedand is relatively unaffected by short-term starvation(Supplemental Fig. S7), diurnal switches, or externalatmospheric conditions, unlike the phloem and seedcoat apoplast, which deliver Suc frommaternal tissues(Geiger and Servaites, 1994; Tuan et al., 1997; Kallarackaland Komor, 1998; Munier-Jolian and Salon, 2003; Kanget al., 2007). Thus, it is the endosperm itself that exertscontrol over the Suc concentration in the endospermvacuole. The amount of Suc stored in the vacuole ishigher than might be predicted on the basis of thedemand of the developing embryo (Zhou et al., 2009)and can support embryo growth for over 20 h if Suc

delivery is cut off. Thus, transient variation in theexternal environment can be buffered to provide thehomeostasis necessary for embryo growth. Suc can besensed by embryo cells (Rolland et al., 2006) and regu-lates both gene expression and embryo development(Iraqi and Tremblay, 2001; Boyer andMcLaughlin, 2004).With Suc acting simultaneously as both a growth sub-strate and a signal molecule, the endosperm is able tosustain the growth of the embryo even in the face oftransient variation in the external environment.

The in Vivo Steady-State Level of Free Amino Acids inthe Endosperm Vacuole

Free amino acids represent the major source ofnitrogen for the growing embryo (Tegeder et al.,2000). Amino acid uptake by the embryo is in part

Figure 8. Spatial expression pattern of Sut1 inendosperm of pea seed and accumulation ofSuc in endosperm of wild-type phenotype andmutant. A, In vivo image corresponds to crosssection through the seed. Expected directionof Suc flow is arrowed. Image is representativefor approximately 30 to 50 mg fresh weight. Bto D, In situ hybridization visualizes local up-regulation of Sut1 within the peripheral re-gions of endosperm (33P labeling appears inblack by bright-field illumination). Nonla-beled tissues are blue colored due to stainingwith toluidine blue. Arrows indicate nonla-beled region of endosperm. E, Amount of Sucaccumulated in endosperm vacuole duringseed development in mutant (mm) and wild-type (Mm) seed. Sucrose content was calcu-lated by volume of endosperm liquid 3 Succoncentration. Arrows indicate temporal up-regulation of SUT-1 in either endosperm orembryo. ch, Chalaza; e, embryo; en, endo-sperm.

Melkus et al.




passive, especially during its early development(DeJong et al., 1997), so that a favorable concentrationgradient needs to be established between the embryoand the endosperm. Our in vivo data have demon-strated the existence of such a gradient. The endo-sperm up-regulates the expression of amino acid

transporters earlier in development and grows morerapidly than does the early embryo, after which itaccumulates Ala and Gln in its vacuole (Fig. 6). Thus,a transient internal source of nitrogen was generatedin the embryo sac, in the form of a liquid mediumenriched with amino acids (Fig. 5C). In this situation,the passive uptake of amino acids by the embryo isencouraged and may indeed represent an essentialcomponent of embryo nutrition during its early de-velopment.

The switch to an active uptake system occurs ratherlate in development and is based on enhanced levels ofAAP1 (an amino acid permease) and PTR1 (a peptidetransporter) in the embryo proper (Miranda et al.,2001). This transport system appears to be controlledby assimilate availability (Bennett and Spanswick,1983). Protein synthesis appears to be nitrogen limited,with the embryo’s nitrogen uptake activity via AAP1being rate limiting for storage protein synthesis(Rolletschek et al., 2005a; Sanders et al., 2009). Here,we have explored the time point at which proteinstorage becomes nitrogen limited and how this relatesto the level of the majority amino acids present in theendosperm vacuole. We have shown that the levels ofAla/Gln gradually increase in the wild-type endo-sperm until the developmental stage reached by a seedof fresh weight 100 to 150 mg. The subsequent declinecoincides temporally with an increase in the rate ofprotein accumulation in the embryo. In contrast, in themutant seeds, the levels of Ala and Gln in the endo-sperm vacuole remain high (Fig. 6, B and C), presum-ably reflecting a much lower demand for amino acidsby the embryo. Our current model suggests that dur-ing early development, when the protein accumula-tion rate and sink strength of embryo are negligible,amino acid demand is exceeded by its supply. How-ever, as soon as protein storage is initiated in theembryo, the balance between demand and supply ofamino acids is altered and an exhaustion of reservesleads to a decline in the amino acid concentration inthe endosperm vacuole. Later, the control of the steady-state amino acid level in the endosperm becomesdemand driven.

A concentration of 60 mM Gln is associated with apeak Gln incorporation rate (Fig. 9C), but the endo-sperm Gln concentration remains below this thresholdduring most of the seed maturation process (Fig. 9B).Thus, protein storage appears to be nitrogen limitedduring the entire main storage phase. The low avail-ability of Gln in the endosperm vacuole is likelyinsufficient to satisfy the requirements of the embryoduring the mid to late developmental stages. Biochem-ical approaches attempting to increase levels of seedprotein, therefore, should focus on increasing the aminoacid content of the endosperm.

The Transient Metabolic Role of the Endosperm

The coordination of seed development clearly re-quires feedback between the filial and the maternal

Figure 9. Steady-state level of metabolites in endosperm vacuole andaccumulation of storage products in embryo during seed development.A, Levels of Suc measured by photometry in endosperm vacuole ofwild-type (wt) and mutant seed and starch accumulation rate in wild-type embryo. B, Levels of Gln measured by HPLC in endospermvacuole of wild-type and mutant seed and protein accumulation rate inwild-type embryo. C, 15N abundance in proteins after in vitro cultiva-tion of wild-type embryos at different levels of Gln in the nutritionmedium. Data points represent means 6 SD of three to five measure-ments. FW, Fresh weight.





tissue (Garcia et al., 2005). The signaling of seed size ina range of species emanates not from the embryo butrather from the endosperm (Felker et al., 1985; Maitzet al., 2000; Garcia et al., 2003). The fact that a retar-dation in embryo growth does not affect seed size inthe pea mutant confirms this notion. The question thatarises is how this control can be metabolically medi-ated. The filial Suc and amino acid pool sizes are clearcandidates as metabolic signals, because these re-spond to altered rates of solute delivery and storageactivity and are known to regulate symporter geneexpression via derepression (Patrick and Offler, 2001).The endosperm is more advanced than the embryowith respect to the accumulation of soluble metabo-lites and the up-regulation of corresponding trans-porters. In the pea mutant, the filial pool is largelyconfined to the endosperm, which accumulates sever-alfold more soluble metabolites than does the wild-typeone. Thus, the endosperm is able to unload nutrientsfrom the seed apoplasm independently of the size ofthe embryo sink. Solute withdrawal from the seedapoplasm can be sensed by a turgor-homeostat mech-anism located in maternal seed tissue, which actsto balance effluxer activity as required (Patrick andOffler, 2001). The growth of the endosperm, therefore,may trigger a wild-type-like feedback signal to thematernal tissue, which could be transmitted via a cal-cium signaling cascade (Zhang et al., 2007) to drivecell elongation in the seed coat of the mutant. Thismechanism would allow the endosperm to retain thecoordination of seed development despite retardedembryo growth. This is attributed to the endosperm-mediatedmetabolic control of seed growth and reflectsthe evolutional predetermination of endosperm: tofavor nutrient transfer from the maternal to the filialgeneration (Sundaresan, 2005).

The frequent observations that the endosperm candrive seed growth and that a retarded endosperm isassociated with reduced embryo growth (Chaudhuryet al., 2001; Choi et al., 2002; Garcia et al., 2003; Ingouffet al., 2006) have been taken to suggest that nutrientsare delivered from the endosperm to the embryo.However, a body of evidence suggests that embryo isautonomous (Weijers et al., 2003; Ungru et al., 2008;Pignocchi et al., 2009) and that metabolite deliveryto the embryo bypasses the bulk endosperm (Yeungand Meinke, 1993; Stadler et al., 2005; Morley-Smithet al., 2008). According to our in vivo data, the extent ofthe trophic subordination of the embryo to the endo-sperm may alter during the course of seed develop-ment. During its early stages, the embryo is physicallyconnected to the maternal seed coat via the suspensor(Supplemental Movies S1 and S2), so it is not possibleto exclude the possibility of this alternative means ofmetabolite delivery to the embryo. Later, following thedestruction of the suspensor, the endosperm blocksany direct contact between the maternal tissue and theembryo, so it must be involved in any metaboliteexchange. The endosperm accumulates Suc and aminoacids in its vacuole (Fig. 6), and these represent the

embryo’s sole source of nutrition. The growth of theembryo is affected when the metabolism of the endo-sperm is compromised (Baud et al., 2005; Ohto et al.,2005; Kondou et al., 2008). This behavior demonstratesthe trophic subordination of the embryo to the endo-sperm. Here, we have shown that the endospermaffords the metabolic environment of the embryo andmediates seed coat growth, at a stage before theembryo’s nutrition can be ensured by its own sinkstrength, and until cell division, tissue differentiation,endopolyploidization, and storage have all been com-pleted. Reciprocal crosses have been shown to expressthe same extent of maternal dominance over thedetermination of seed size (Lemontey et al., 2000).

Proteomic and transcriptomic analyses in M. trun-catula have indicated that, as the embryo acquires itsown sink, so the endosperm’s metabolism shifts from ahighly active to a quiescent state (Gallardo et al., 2007).At this stage, the transient nature of storage in thedicotyledonous endosperm is crucial: the endospermdeposits soluble metabolites, which are neither pre-served nor returned to the maternal organism. Instead,the embryo utilizes these reserves, and, as it grows, ittakes over the physical space previously occupied bythe endosperm. A transient endosperm, therefore,allows the embryo to avoid competition for nutritionand space within the maternal organism, which couldotherwise restrict embryo growth under stressful con-ditions or affect gene expression as a result of me-chanical pressure (Weijers et al., 2003; Desprat et al.,2008). Therefore, the endosperm largely accomplishesits function before seed maturation. Its job complete,the liquid endosperm then disappears. As Schillerputs it in his play The Genoese Conspiracy, “the Moorhas done his duty, the Moor can go” (Schiller, 1799).

MATERIALS AND METHODS

Plant Material

Pea (Pisum sativum ‘Erbi’) and E2748 mutant (Johnson et al., 1994) plants

were grown under a 16-h-light, 19�C/8-h-dark, 16�C regime. Because homo-

zygous E2748 mutant seeds are lethal, plants were produced by embryo

rescue. For this purpose, the developing embryo of a homozygous mutant

seed (identified by phenotype, following Johnson et al. [1994]) was hand

dissected, germinated, and grown under sterile conditions until a shoot was

formed, at which point the seedling was transferred to soil. The wild-type

phenotype was restored by transferring pollen from a wild-type plant.

Sampling and Biochemical Analysis

Endosperm liquid was isolated by microsyringe (two endosperm samples

per individual seed). Set volumes (3–10 mL) were added to 200 mL of 80%

ethanol and processed as described elsewhere (Borisjuk et al., 2002). Free

amino acids weremeasured by HPLC, and soluble sugars weremeasured by a

coupled photometric assay (Rolletschek et al., 2005a, 2005b). Starch was

measured enzymatically (Rolletschek et al., 2002). Total nitrogen was assessed

from powdered seed samples (dried overnight at 70�C) by elemental analysis

(Rolletschek et al., 2002). Crude protein was calculated from total nitrogen 36.25. Accumulation rates were calculated from measured starch/protein

contents and the embryo weight. Fresh and dry weight accumulation rates

were derived by weighing the seed before drying and removing the embryos

before reweighing.

Melkus et al.




Immunoassay

Seeds were fixed in either 2.5% (v/v) glutaraldehyde, 50 mM sodium

cacodylate buffer (pH 7) or 4% (w/v) paraformaldehyde, 50 mM potassium

phosphate buffer (pH 7) under a mild vacuum for 4 h at room temperature,

rinsed in cacodylate buffer, dehydrated, and embedded in butyl-methyl

methacrylate by polymerization at 20�C for 48 h under UV light. Sections of

3 to 5 mm thickness were cut on a microtome. The immunolocalization

procedure was performed using an affinity-purified anti-legumin polyclonal

antibody and the corresponding VASTASTAIN ABC-AP kit (Alkaline Phos-

phatase Substrate Kit III) and evaluated microscopically.

Electron Microscopy

Seed sections were immersed in 2% (w/v) paraformaldehyde, 0.5% (v/v)

glutaraldehyde in 50 mM potassium phosphate buffer, 5 mM EGTA (pH 7.2),

5 mM CaCl2, and 3% (w/v) Suc. After 2 h at room temperature, fixation was

continued overnight on ice using freshly prepared solution. All subsequent

steps were performed as described elsewhere (Borisjuk et al., 2002).

Laser Microdissection, RT-PCR Analysis, and in

Situ Hybridization

Seeds were frozen at 280�C, cut into 10-mm sections at 220�C, mounted

onto PET membrane-covering glass slides (Carl Zeiss MicroImaging), and

lyophilized at 220�C. The sections were subjected to laser-assisted microdis-

section using a PALM Laser-Microbeam (Bernried) device. Small tissue

samples were collected using laser pressure catapulting and larger ones by

a hand-operatedmicroneedle. Poly(A) mRNAwas extracted directly using the

Dynabeads mRNA DIRECT kit (Dynal Biotech) using the manufacturer’s

protocol. Linear amplification and cDNA synthesis were carried out from

approximately 1 ng of mRNA using the ExpressArt mRNA Amplification

Nano kit (AmpTec) according to the manufacturer’s protocol. PCR was based

on a template of 20 ng of cDNA in a 25-mL volume containing 0.2 mM gene-

specific primers, 200 mM desoxyribonucleotide triphosphate (Pharmacia),

0.5 units of Taq polymerase (Roche), and 13 polymerase buffer supplied

with the enzyme. The primer sequences used and predicted amplicon sizes

are given in Supplemental Table S2. The PCR regime consisted of a 94�Cincubation for 2 min followed by 30 cycles of 94�C for 30 s, 57�C for 30 s, and

72�C for 60 s. PCR products were separated by 0.8% agarose gel electropho-

resis and visualized by ethidium bromide staining.

cDNA labeling and in situ hybridization were carried out as described by

Borisjuk et al. (2002). The complete cDNA of Vicia faba SUT1 (Weber et al.,

1997) was used as probe after labeling with [33P]dCTP.

In Vitro Incubation of Pea Embryos

Freshly isolated, intact embryos were transferred into 25-mL vials con-

taining an incubation buffer consisting of 250 mM Suc, 80 mM Ala, 20 mM KCl,

4 mM CaCl2, 2 mM K2SO4, 2 mM KH2PO4, 3 mM MgSO4 and 10 mM MES/KOH

(pH 5.6). Subsequently, 15N-labeled Gln (Campro Scientific) was added to

reach final concentrations of 10, 30, 60, 90, 120, and 150 mM. Osmolality was

held constant by the addition of mannitol. The vials were incubated in light

(25 mE) with gentle agitation. After 20 h, the embryos were rinsed twice in

distilled water and immediately frozen. The protein faction was extracted, and

the 14N/15N isotope pair was analyzed using elemental analysis coupled to

isotope ratio mass spectrometry (Borisjuk et al., 2007).

NMR Protocol

Instrument

The experiments were performed on a 17.6-T wide-bore (89 mm) super-

conducting magnet (Bruker BioSpin) equippedwith actively shielded imaging

gradients. A 200 mT m21 gradient system and a 15-mm birdcage resonator

were used for the identification and quantification of metabolites. High spatial

resolution spectroscopic imaging experiments were performed with a 1 T m21

gradient system and a 20-mm birdcage coil.

Plant Material

For high-resolution metabolite imaging, seeds were detached from the pod

and immediately immersed in buffer (as used for in vitro incubation of

embryos but lacking Suc, Ala, and Gln). For all other experiments, the seed

was detached from the pod and presented to the instrument within minutes.

During the period of measurement, the seed was kept in the dark at 20�C and

ambient atmosphere.

Metabolite Identification

Localized one-dimensional PRESS (repetition time [TR] = 2 s, echo time

[TE] = 20 ms, voxel = 1 mm3, no. of averages [NA] = 32) and L-COSY (Thomas

et al., 2001; TR = 2 s, TE = 10 ms, voxel = 1.5 mm3, acquired points in the

indirect spectroscopic direction [t1] increments = 1,024, NA= 1) were used for

metabolite identification.

T1 Measurement, Quantification, and Metabolite Mapping

T1 measurements were performed by extending the CSI sequence with a

nonselective adiabatic inversion pulse (hyperbolic secant pulse; 3 ms), a

variable inversion time (TI) before signal excitation, and a 10-s recovery delay

after signal acquisition. The experiments were performed for five different TIs

(nonlinearly distributed between 98 and 5,000 ms). To avoid side-lobe effects

from the spatial response function and contamination from surrounding

areas, an acquisition-weighted k-space sampling (hanning) was applied

(Pohmann and von Kienlin, 2001). The parameters for the T1 CSI protocol

were as follows: TR = 10 s, number of total scans (NS) = 100, field of view

(FOV) = 8 3 8 mm2, spatial resolution = 1 3 1 mm2 in plane, 0.2-mm slice

thickness, spectral = 1,024 points, bandwidth = 8 kHz. The VAPOR (for

variable pulse power and optimized relaxation delays) suppression scheme

using frequency-selective hermite pulses of 300 Hz bandwidth (Tkac et al.,

1999) was used to ensure water suppression. The T1 values for the metabolite

resonances at various developmental stages are given in Supplemental Table

S1. A low-spatial-resolution CSI method with a long repetition time (TR = 10

s), NS = 200 (hanning weighted), FOV = 8 3 8 mm2, spatial resolution = 0.8 30.8 mm2 in plane, 1-mm slice thickness, spectral = 2,048 points, bandwidth = 8

kHz, acquisition delay = 1 ms, VAPOR water suppression (hermite pulses;

bandwith = 200–600 Hz) was employed to quantify metabolites in the

endosperm. The duration of the spectroscopic imaging experiment was 33

min, 20 s. The high-spatial-resolution CSI experiment was performed with a

1 T m21 gradient system, using the following experimental parameters: TR =

1.5 s, NS = 27,500 (hanning weighted), FOV = 5 3 5 mm2, spatial resolution =

100 3 100 mm2 in plane, 100-mm slice thickness, spectral = 2,048 points,

bandwidth = 8 kHz, acquisition delay = 2 ms, VAPOR water suppression

(hermite pulses; bandwith = 500 Hz). The total duration for the spectroscopic

imaging experiment was 11 h, 27 min.

Single Voxel Spectroscopy

Changes in metabolite concentration in the pea endosperm were observed

over 6 h using voxel-selective PRESS (TR = 2 s, TE = 20 ms, voxel size = 1 mm3,

NA = 128). The experiments were performed under nitrogen or oxygen

(premixed gas bottles; purity of 99.999%) by aerating the seeds via a gas tube

mounted within the NMR instrument. This treatment induced almost imme-

diate changes in the oxygen level inside the coil, as checked by oxygen

microsensors (Presens; for details, see Rolletschek et al., 2005b). An oxygen

level of less than 2 mM is regarded as anoxic conditions.

Morphological Imaging

The structural and morphological imaging of the seeds was achieved using

gradient-echo multislice images (FLASH) with the following parameters: TR =

500 ms, TE = 30ms, FOV = 103 10mm2, matrix = 1282, slice thickness = 0.5 mm,

NA = 4, total acquisition time = 1 min, 42 s. Three-dimensional data sets were

acquired using a three-dimensional spin-echo sequence with TR = 1 s, TE = 7.7

ms, FOV = 203 7.53 7.5 mm3, matrix = 2563 963 96, isotropic resolution = 78

mm3, total measurement time = 2 h, 33 min for a 120-mg seed and TR = 1 s, TE =

6.9 ms, FOV = 203 73 7 mm3, matrix = 3363 1163 116, isotropic resolution =

60 mm3, total measurement time = 3 h, 44 min for 30- and 73-mg seeds.

Postprocessing and Quantification

To estimate the metabolite concentration in the endosperm, an external

5-mm-diameter reference tube containing 50 mM aqueous Suc, Glc, Gln, or Ala





wasmeasured by the same spectroscopic imaging protocol used for the in vivo

measurements. Signal correction between reference and in vivo samples was

based on Soher et al. (1996) and DeGraaf (2007). The long repetition time (TR =

10 s) of the CSI experiment enabled sufficient longitudinal spin relaxation, so

correction factors for partial saturation were not required. The repetition time

was more than 5-fold the longest metabolite T1 (Supplemental Table S1). No

B1 field correction was applied, because the 15-mm birdcage resonator had a

homogenous B1 distribution in phantom measurements. The in vivo metab-

olite concentration (KI) was calculated from the known external reference

metabolite concentration (KR) using:

KI ¼ KR � SISR

� CLOAD

where SI and SR are the in vivo (I) and reference (R) signal amplitudes acquired

with the chemical shift imaging method. The correction factor for the different

coil loading between the in vivo seed and reference (CLOAD) was involved in

the quantification process (Soher et al., 1996). CLOAD can be estimated from the

difference in the radio frequency (RF) power:

CLOAD ¼ 10DA=20

where DA is the difference in power attenuation for the 90� pulse between the

two experiments, measured in decibels. The accuracy of CLOAD was checked

using eight water phantoms with different solute fractions of sodium chloride

(0.1%–20%). The attenuation required to achieve a 90� pulse and the water

signal amplitude were measured for each phantom.

Data Processing

Raw spectroscopic imaging data were preprocessed using an in-house

computer program written in MATLAB 7.0.1 (MathWorks). The NMR signal

was analyzed with the interactive time domain fit algorithm AMARES

(Vanhamme et al., 1997) within the jMRUI software package (Naressi et al.,

2001).

MRI Calibration

To ensure the reliability of the method, an external calibration procedure

was applied. Individual seeds at selected developmental stages were analyzed

under identical experimental conditions. Immediately after the MRI, endo-

sperm samples were isolated by microsyringe, their free amino acid content

was derived by HPLC, and their soluble sugar content was derived by

photometry. The MRI signals were plotted against the measured metabolite

concentrations.

Sequence data from this article can be found in the GenBank/EMBL

data libraries under accession numbers AY956395, AY956396, DQ221698,

AF109922, and X90378.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Phenotype of mutant E2748.

Supplemental Figure S2. Distribution of storage product accumulation in

embryo versus endosperm in mutant seed.

Supplemental Figure S3. Schematic representation of the L-COSY pulse

sequence (Thomas et al., 2001) for the application on pea seed.

Supplemental Figure S4. Localized PRESS spectra from different regions

of the endosperm of wild-type seed (approximately 120 mg fresh

weight)

Supplemental Figure S5. Localized CSI spectra from the endosperm of

wild-type seeds at two different developmental stages.

Supplemental Figure S6. Correlation of noninvasive measurement with

data derived from conventional assays in dissected samples of endo-

sperm.

Supplemental Figure S7. Compositional changes of liquid endosperm

during the starvation experiment measured by NMR.

Supplemental Table S1. T1 relaxation times of metabolites in the pea

endosperm at 17.6 T.

Supplemental Table S2. Primers used in RT-PCR analyses.

Supplemental Movie S1. Internal structure of seed by three-dimensional

NMR imaging (corresponds to Fig. 1, D and G).


NMR imaging (corresponds to Fig. 1, E and H).


NMR imaging (corresponds to Fig. 1, F and I).

Supplemental Movie S4.Digital model of three-dimensional anatomy of a

pea seed (corresponds to Fig. 2B).

ACKNOWLEDGMENTS

We are grateful to A. Webb and T. Neuberger for their help with and

discussion about MRI and to M. Flentje for his cooperation. We thank T.L.

Wang for providing seed of the E2748 mutant. Special thanks to A. Schwarz,

K. Blaschek, and A. Stegmann for their excellent technical assistance and to

U. Tiemann and K. Lipfert for artwork.

Received June 30, 2009; accepted September 8, 2009; published September 11,

2009.

LITERATURE CITED

Bate NJ, Niu X, Wang Y, Reimann KS, Helentjaris TG (2004) An invertase

inhibitor from maize localizes to the embryo surrounding region during

early kernel development. Plant Physiol 134: 246–254

Baud S, Wuilleme S, Lemoine R, Kronenberger J, Caboche M, Lepiniec L,

Rochat C (2005) The AtSUC5 sucrose transporter specifically expressed

in the endosperm is involved in early seed development in Arabidopsis.

Plant J 43: 824–836

Bennett AB, Spanswick RM (1983) Derepression of amino acid-H+ co-

transport in developing soybean embryos. Plant Physiol 72: 781–786

Berger F, Grini PE, Schnittger A (2006) Endosperm: an integrator of seed

growth and development. Curr Opin Plant Biol 9: 664–670

Bewley JD, Black M (1994) Seeds: Physiology of Development and Ger-

mination, Ed 2. Plenum Press, New York

Boisnard-Lorig C, Colon-Carmona A, Bauch M, Hodge S, Doerner P,

Bancharel E, Dumas C, Haseloff J, Berger F (2001) Dynamic analyses of

the expression of the HISTONE:YFP fusion protein in Arabidopsis show

that syncytial endosperm is divided in mitotic domains. Plant Cell 13:

495–509

Borisjuk L, Macherel D, Benamar A, Wobus U, Rolletschek H (2007) Low

oxygen sensing and balancing in plant seeds: a role for nitric oxide. New

Phytol 176: 813–823

Borisjuk L, Rolletschek H (2009) The oxygen status of the developing seed.

New Phytol 182: 17–30

Borisjuk L, Rolletschek H, Wobus U, Weber H (2003) Differentiation of

legume cotyledons as related to metabolic gradients and assimilate

transport into seeds. J Exp Bot 54: 503–512

Borisjuk L, Wang TL, Rolletschek H, Wobus U, Weber H (2002) A pea seed

mutant affected in the differentiation of the embryonic epidermis is

impaired in embryo growth and seed maturation. Development 129:

1595–1607

Bottomley PA (1984) Selective volume method for performing localized

NMR spectroscopy. U.S. Patent No. 4 480 228

Bourgeois D, Remy C, Lefur Y, Devoulon P, Benabid AL, Decorps M

(1991) Proton spectroscopic imaging: a tool for studying intracerebral

tumor models in rat. Magn Reson Med 21: 10–20

Boyer JS, McLaughlin JE (2004) Sugar-responsive gene expression, inver-

tase activity, and senescence in aborting maize ovaries at low water

potentials. Ann Bot (Lond) 94: 675–689

Chaudhury AM, Koltunow A, Payne T, Luo M, Tucker MR, Ming L,

Dennis E, Peacock WJ (2001) Control of early seed development. Annu

Rev Cell Dev Biol 17: 677–699

Melkus et al.




Chiou TJ, Bush DR (1998) Sucrose is a signal molecule in assimilate

partitioning. Proc Natl Acad Sci USA 95: 4784–4788

Choi Y, Gehring M, Johnson L, Hannon M, Harada JJ, Goldberg RB,

Jacobsen SE, Fischer RL (2002) DEMETER, a DNA glycosylase domain

protein, is required for endosperm gene imprinting and seed viability in

Arabidopsis. Cell 110: 33–42

DeGraaf R (2007) In Vivo NMR Spectroscopy: Principles and Techniques,

Ed 2. John Wiley & Sons, Chichester, UK

DeJong A, Koerselman-Kooij JW, Schuurmans JAMJ, Borstlap AC (1997)

The mechanisms of amino acid efflux from seed coats of developing pea

seeds as revealed by uptake experiments. Plant Physiol 114: 731–736

Desprat N, Supatto W, Pouille P, Beaurepaire E, Farge E (2008) Tissue

deformation modulates twist expression to determine anterior midgut

differentiation in Drosophila embryos. Dev Cell 15: 470–477

Felker FC, Peterson DM, Nelson OE (1985) Anatomy of immature grains of

eight maternal effect shrunken endosperm barley mutants. Am J Bot 72:

248–256

Gallardo K, Firnhaber C, Zuber H, Hericher D, Belghazi M, Henry C,

Kuster H, Thompson R (2007) A combined proteome and transcriptome

analysis of developingMedicago truncatula seeds: evidence for metabolic

specialization of maternal and filial tissues. Mol Cell Proteomics 6:

2165–2179

Garcia D, FitzGerald JN, Berger F (2005) Maternal control of integument

cell elongation and zygotic control of endosperm growth are coordi-

nated to determine seed size in Arabidopsis. Plant Cell 17: 52–60

Garcia D, Saingery V, Chambrier P, Mayer U, Jurgens G, Berger F (2003)

Arabidopsis haiku mutants reveal new controls of seed size by endo-

sperm. Plant Physiol 131: 1661–1670

Gehring M, Choi Y, Fischer RL (2004) Imprinting and seed development.

Plant Cell 16: 203–213

Geiger DR, Servaites JC (1994) Diurnal regulation of photosynthetic

carbon metabolism in C-3 plants. Annu Rev Plant Physiol 45: 235–256

Gifford RM, Thorne JH (1985) Sucrose concentration at the apoplastic

interface between seed coat and cotyledons of developing soybean

seeds. Plant Physiol 77: 863–868

Haig D, Westoby M (2006) An earlier formulation of the genetic conflict

hypothesis of genomic imprinting. Nat Genet 38: 271

Hauxwell AJ, Corke FMK, Hedley CW, Wang TL (1990) Storage protein

gene expression is localized to regions lacking mitotic activity in

developing pea embryos: an analysis of seed development in Pisum

sativum. Development 110: 283–289

Hill LM, Morley-Smith ER, Rawsthorne S (2003) Metabolism of sugars in

the endosperm of developing seeds of oilseed rape. Plant Physiol 131:

228–236

Ingouff M, Jullien PE, Berger F (2006) The female gametophyte and the

endosperm control cell proliferation and differentiation of the seed coat

in Arabidopsis. Plant Cell 18: 3491–3501

Iraqi D, Tremblay FM (2001) Analysis of carbohydrate metabolism en-

zymes and cellular contents of sugars and proteins during spruce

somatic embryogenesis suggests a regulatory role of exogenous sucrose

in embryo development. J Exp Bot 52: 2301–2311

Johnson S, Liu CM, Hedley CL, Wang TL (1994) An analysis of seed

development in Pisum sativum. XVIII. The isolation of mutants defective

in embryo development. J Exp Bot 45: 1503–1511

Kallarackal J, Komor E (1998) Transport of hexoses by phloem of Ricinus

commuis L. seedlings. Planta 177: 336–341

Kang Y, Outlaw WH Jr, Andersen PC, Fiore GB (2007) Guard-cell apo-

plastic sucrose concentration: a link between leaf photosynthesis and

stomatal aperture size in the apoplastic phloem loader Vicia faba L. Plant

Cell Environ 30: 551–558

Kockenberger W (2001) Nuclear magnetic resonance micro-imaging in the

investigation of plant cell metabolism. J Exp Bot 52: 641–652

Kockenberger W, DePanfilis C, Santoro D, Dahiva P, Rawsthorne S (2004)

High resolution NMR microscopy of plants and fungi. J Microsc 214:

182–189

Kondou Y, Nakazawa M, Kawashima M, Ichikawa T, Yoshizumi T,

Kumiko S, Ishikawa A, Koshi T, Matsui R, Muto S, et al (2008)

RETARDED GROWTH OF EMBRYO1, a new basic helix-loop-helix

protein, expresses in endosperm to control embryo growth. Plant

Physiol 147: 1924–1935

Lemontey C, Mousset-Declas C, Munier-Jolain N, Boutin JP (2000)

Maternal genotype influences pea seed size by controlling both mitotic

activity during early embryogenesis and final endoreduplication level/

cotyledon cell size in mature seed. J Exp Bot 51: 167–175

Lersten NR (2004) Endosperm. In Flowering Plant Embryology: With

Emphasis on Economic Species. Wiley-Blackwell, Oxford, pp 150–169

Looger LL, Lalonde S, Frommer WB (2005) Genetically encoded FRET

sensors for visualizing metabolites with subcellular resolution in living

cells. Plant Physiol 138: 555–557

Maitz M, Santandrea G, Zhang ZY, Lal S, Hannah LC, Salamini F,

Thompson RD (2000) Rgf1, a mutation reducing grain filling in maize

through effects on basal endosperm and pedicel development. Plant J

23: 29–42

Marinos NG (1970) Embryogenesis of the pea (Pisum sativum). I. The

cytological environment of the developing embryo. Protoplasma 70:

261–279

Metzler A, Izquierdo M, Ziegler A, Kockenberger W, Komor E, von

Kienlin M, Haase A, Decorps M (1995) Plant histochemistry by corre-

lation peak imaging. Proc Natl Acad Sci USA 92: 11912–11915

Miranda M, Borisjuk L, Tewes A, Heim U, Sauer N, Wobus U, Weber H

(2001) Amino acid permeases in developing seeds of Vicia faba L.:

expression precedes storage protein synthesis and is regulated by amino

acid supply. Plant J 28: 61–71

Morley-Smith ER, Pike MJ, Findlay K, Kockenberger W, Hill LM, Smith

AM, Rawsthorne S (2008) The transport of sugars to developing

embryos is not via the bulk endosperm in oilseed rape seeds. Plant

Physiol 147: 2121–2130

Munier-Jolian N, Salon C (2003) Can sucrose content in the phloem sap

reaching field pea seeds (Pisum sativum L.) be an accurate indicator of

seed growth potential? J Exp Bot 54: 2457–2465

Murray DR (1987) Nutritive role of seed coats in developing legume seeds.

Am J Bot 74: 1122–1137

Naressi A, Couturier C, Devos JM, Janssen M, Mangeat C, de Beer R,

Graveron-Demilly D (2001) MRUI, MRUI for Java. MAGMA 12:

141–152

Neuberger T, Sreenivasulu N, Rokitta M, Rolletschek H, Gobel C, Rutten

T, Radchuk V, Feussner I, Wobus U, Jakob P, et al (2008) Quantitative

imaging of oil storage in developing crop seeds. Plant Biotechnol J 6:

31–45

Neuberger T, Webb A (2009) Radiofrequency coils for magnetic resonance

microscopy. NMR Biomed (in press)

Nowack MK, Shirzadi R, Dissmeyer N, Dolf A, Endl E, Grini PE,

Schnittger A (2007) Bypassing genomic imprinting allows seed devel-

opment. Nature 447: 312–315

Ohto MA, Fischer RL, Goldberg RB, Nakamura K, Harada JJ (2005)

Control of seed mass by APETALA2. Proc Natl Acad Sci USA 102:

3123–3128

Olsen OA (2004) Nuclear endosperm development in cereals and Arabi-

dopsis thaliana. Plant Cell (Suppl) 16: S214–S227

Patrick JW, Offler CE (2001) Compartmentation of transport and transfer

events in developing seeds. J Exp Bot 52: 551–564

Pignocchi C, Minns GE, Nesi N, Koumproglou R, Kitsios G, Benning C,

LloydWC, Doonan JH, Hills MJ (2009) ENDOSPERMDEFECTIVE1 is a

novel microtubule-associated protein essential for seed development in

Arabidopsis. Plant Cell 21: 90–105

Pohmann R, von Kienlin M (2001) Accurate phosphorus metabolite

images of the human heart by 3D acquisition-weighted CSI. Magn

Reson Med 45: 817–826

Ratcliffe RG, Shachar-Hill Y (2001) Probing plant metabolism with NMR.

Annu Rev Plant Physiol Plant Mol Biol 52: 499–526

Rolland F, Baena-Gonzalez E, Sheen J (2006) Sugar sensing and signaling

in plants: conserved and novel mechanisms. Annu Rev Plant Biol 57:

675–709

Rolletschek H, Hajirezaei M, Wobus U, Weber H (2002) Antisense-inhibition

of ADP-glucose pyrophosphorylase in Vicia narbonensis seeds increases

soluble sugars and lead to higher water and nitrogen uptake. Planta 214:

954–964

Rolletschek H, Hosein F, Miranda M, Heim U, Gotz KP, Schlereth A,

Borisjuk L, Saalbach I, Wobus U, Weber H (2005a) Ectopic expression

of an amino acid transporter (VfAAP1) in seeds of Vicia narbonensis and

Pisum sativum increases storage proteins. Plant Physiol 137: 1236–1249

Rolletschek H, Koch K, Wobus U, Borisjuk L (2005b) Positional cues for

the starch/lipid balance in maize kernels and resource partitioning to

the embryo. Plant J 42: 69–83

Rosche E, Blackmore D, Tegeder M, Richardson T, Schroeder H, Higgins





TJV, Frommer WB, Offler CE, Patrick JW (2002) Seed-specific over-

expression of a potato sucrose transporter increases sucrose uptake and

growth rates of developing pea cotyledons. Plant J 30: 165–175

Sabelli PA, Larkins BA (2009) The development of endosperm in grasses.

Plant Physiol 149: 14–26

Sanders A, Collier R, Trethewy A, Gould G, Sieker R, Tegeder M (2009)

AAP1 regulates import of amino acids into developing Arabidopsis

embryos. Plant Physiol 59: 540–552

Schiller F (1799) Fiesco or the Genoese Conspiracy: A Tragedy. J Archer.

2004 reproduction of original from the British Library. Thomson Gale,

Farmington Hills, MI

Schneider B, Holscher D (2007) Laser microdissection and cryogenic

nuclear magnetic resonance spectroscopy: an alliance for cell type-

specific metabolite profiling. Planta 225: 763–770

Soher BJ, van Zijl PC, Duyn JH, Barker PB (1996) Quantitative proton MR

spectroscopic imaging of the human brain. Magn Reson Med 35:

356–363

Stadler R, Lauterbach C, Sauer N (2005) Cell-to-cell movement of green

fluorescent protein reveals post-phloem transport in the outer integu-

ment and identifies symplastic domains in Arabidopsis seeds and

embryos. Plant Physiol 139: 701–712

Stewart-Cox JA, Britton NF, Mogie M (2004) Endosperm triploidy has a

selective advantage during ongoing parental conflict by imprinting.

Proc Biol Sci 271: 1737–1743

Sundaresan V (2005) Control of seed size in plants. Proc Natl Acad Sci USA

102: 17887–17888

Supena ED, Winarto B, Riksen T, Dubas E, van Lammeren A, Offringa R,

Boutilier K, Custers J (2008) Regeneration of zygotic-like microspore-

derived embryos suggests an important role for the suspensor in early

embryo patterning. J Exp Bot 59: 803–814

Tanaka H, Onouchi H, Kondo M, Hara-Nishimura I, Nishimura M,

Machida C, Machida Y (2001) A subtilisin-like serine protease is

required for epidermal surface formation in Arabidopsis embryos and

juvenile plants. Development 128: 4681–4689

Tegeder M, Offler CE, Frommer WB, Patrick JW (2000) Amino acid

transporters are localized to transfer cells of developing pea seeds.

Plant Physiol 122: 319–326

Tegeder M, Wang XD, Frommer WB, Offler CE, Patrick JW (1999) Sucrose

transport into developing seeds of Pisum sativum L. Plant J 18: 151–161

Thomas MA, Yue K, Binesh N (2001) Localized two-dimensional shift

correlated MR spectroscopy of human brain. Magn Reson Med 46:

58–67

Tkac I, Starcuk Z, Choi IY, Gruetter R (1999) In vivo 1H NMR spectroscopy

of rat brain at ms echo time. Magn Reson Med 41: 649–656

Tse TY, Spanswick RM, Jelinski LW (1996) Quantitative evaluation of

NMR and MRI methods to measure sucrose concentrations in plants.

Protoplasma 194: 54–62

Tuan YL, Patrick JW, Brady CJ (1997) The composition of apoplast fluid

recovered from intact developing tomato fruit. Aust J Plant Physiol

23: 9–13

Ungru A, Nowack MK, Reymond M, Shirzadi R, Kumar M, Biewers S,

Grini PE, Schnittger A (2008) Natural variation in the degree of

autonomous endosperm formation reveals independence and con-

straints of embryo growth during seed development in Arabidopsis

thaliana. Genetics 179: 829–841

Vanhamme L, van den Boogaart A, Van Huffel S (1997) Improved method

for accurate and efficient quantification of MRS data with use of prior

knowledge. J Magn Reson 129: 35–43

Weber H, Borisjuk L, Heim U, Sauer N, Wobus U (1997) A role for sugar

transporters during seed development: molecular characterization

of a hexose and a sucrose carrier in fava bean seeds. Plant Cell 9:

895–908

Weijers D, van Hamburg JP, van Rijn E, Hooykaas PJJ, Offringa R (2003)

Diphtheria toxin-mediated cell ablation reveals interregional commu-

nication during Arabidopsis seed development. Plant Physiol 133:

1882–1892

Yeung EC, Meinke DW (1993) Embryogenesis in angiosperms: develop-

ment of the suspensor. Plant Cell 5: 1371–1381

Zhang WH, Zhou Y, Dibley KE, Tyerman SD, Furbank RT, Patrick JW

(2007) Nutrient loading of developing seeds. Funct Plant Biol 34:

314–331

Zhou Y, Chan K, Wang TL, Hedley CL, Offler CE, Patrick JW (2009)

Intracellular sucrose communicates metabolic demand to sucrose trans-

porters in developing pea cotyledons. J Exp Bot 60: 71–85

Melkus et al.




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