The Metabolic Role of the Legume Endosperm: A Noninvasive Imaging Study 1[W][OA] Gerd Melkus 2 , Hardy Rolletschek 2 , Ruslana Radchuk, Johannes Fuchs, Twan Rutten, Ulrich Wobus, Thomas Altmann, Peter Jakob, and Ljudmilla Borisjuk* University of Wu ¨rzburg, Institute of Experimental Physics 5, 97074 Wuerzburg, Germany (G.M., J.F., P.J.); and Leibniz-Institut fu ¨ r 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. We designed noninvasive nuclear magnetic resonance tools for the in vivo study of key metabolites in the transient liquid endosperm of intact pea (Pisum sativum) seeds. The steady-state levels of sucrose, glutamine, and alanine could be monitored and 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 laser microdissection, isotope labeling, in situ hybridization, and electron microscopy, was used to contrast the wild-type endosperm with that of a mutant in which embryo growth is retarded. Expression of sequences encoding amino acid and sucrose transporters was up-regulated earlier in the endosperm than in the embryo, and this activity led to the accumulation of soluble metabolites in the endosperm vacuole. The endosperm provides a temporary source of nutrition, permits space for embryo growth, and acts as a buffer between the maternal organism and its offspring. The concentration of sucrose in the endosperm 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 that the young embryo develops within a homeostatic environment, necessary to sustain embryogenesis. We argue for a degree of metabolite-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 from maternal 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 fertilization event (Bewley and Black, 1994), but the developmental fates of these two organs differ greatly from one another. The embryo progresses through a fixed pat- tern of cellular divisions to form a series of specialized organs, whereas the endosperm, which initially grows coenocytically, either becomes a major storage organ (monocotyledonous species) or degenerates (dicotyle- donous species) as the seed matures (Olsen, 2004; Sabelli and Larkins, 2009). The monocotyledonous endosperm serves as a crucial provider of nutrition during germination, while the role of the transient dicotyledonous endosperm has remained a puzzle despite 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 series of genetic and epigenetic interactions in the endosperm is now known to play a crucial regulatory role over embryogenesis (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 of maternal over paternal chromosomes, the mother may be able to exert greater control over nutrient allo- cation to the progeny and, in so doing, maximize her own fitness. The triploid state of endosperm appears to be advantageous for the transfer of nutrients from the maternal to the filial generation (Stewart-Cox et al., 2004; Sundaresan, 2005). Many genes/transcription factors active in the endosperm have been identified as having an effect on seed size (Ohto et al., 2005; Kondou et al., 2008), whereas the embryo contributes comparatively little to this trait (Garcia et al., 2005; Ingouff et al., 2006). The growth of the seed relies on a degree of coordination exercised by the endosperm, but how this is achieved at the metabolic level remains unclear. The small seed size of the model plants Arabidopsis (Arabidopsis thaliana) and Medicago truncatula presents a technical challenge for the analysis of endosperm composition. The application of laser microdissection 1 This work was supported by the Federal Ministry of Education and Research. 2 These authors contributed equally to the article. * Corresponding author; e-mail [email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described 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- scription. www.plantphysiol.org/cgi/doi/10.1104/pp.109.143974 Plant Physiology Ò , November 2009, Vol. 151, pp. 1139–1154, www.plantphysiol.org Ó 2009 American Society of Plant Biologists 1139 https://plantphysiol.org Downloaded on November 4, 2020. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
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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-
(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.
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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.
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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.
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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.
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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.
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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.
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