Abortive seed development in Ulmus minor (Ulmaceae)

Botanical Journal of the Linnean Society

, 2004,

145

, 455–467. With 29 figures

© 2004 The Linnean Society of London,

Botanical Journal of the Linnean Society,

2004,

145

, 455–467

455

Blackwell Science, LtdOxford, UKBOJBotanical Journal of the Linnean Society0024-4074The Linnean Society of London, 2004? 2004145?455467Original Article

ABORTIVE SEEDS IN ULMUS MINORJ. C. LÓPEZ-ALMANSA ET AL.

*Corresponding author. E-mail: [email protected]†Current address: Universidad Católica de Ávila, C/Canteros s/n, 05005 Ávila, Spain.

Abortive seed development in

Ulmus minor

(Ulmaceae)

JUAN CARLOS LÓPEZ-ALMANSA

1†

, EDWARD C. YEUNG

2

and LUIS GIL

1

*

1

Unidad de Anatomía, Fisiología y Mejora Genética, E. T. S. Ingenieros de Montes, Universidad Politécnica de Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain

2

Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1 N4

Received May 2003; accepted for publication February 2004

Three genotypes of field elm (

Ulmus minor

) were studied to determine the structural basis of seed abortion in thisspecies. In the non-abortive control, P-VV1, the pattern of seed development is similar to many flowering plants. Theembryo progresses through defined morphological stages leading to developmental arrest as the seed matures. Stor-age products are abundant within embryo cells. Endosperm development is similar to the nuclear type; however, amore extensive cellularization of the endosperm occurs prior to it being crushed by the expanding embryo. For theabortive genotypes, M-SF1 and V-JR1, abnormalities in endosperm development are found. This is judged by theearly cellularization and the massive synthesis of the PAS-positive material in the cellular endosperm. In these abor-tive genotypes, embryo development is delayed and storage products failed to accumulate within embryo cells. Afterseed desiccation, no living embryo tissue remains within the seed coat in the abortive genotypes. © 2004 The Lin-nean Society of London,

Botanical Journal of the Linnean Society

, 2004,

145

, 455–467.

ADDITIONAL KEYWORDS:

elm – endosperm – plant embryology – seed abortion – zygotic embryo.

INTRODUCTION

Field elm,

Ulmus minor

Mill., is a tree of great ecolog-ical and cultural importance in Europe. Humans havemade use of this tree since prehistoric times as asource of timber, foliage and other ornamental uses(Richens, 1983; Heybroek, 1990). The land once occu-pied by elms has been gradually converted into culti-vated lands. As a result, there has been a reduction inthe natural elm population. The population of elm hasbeen further eroded by the infection of a pathogenicvascular fungus,

Ophiostoma ulmi

(Buism.) Nannf.This results in the well-known Dutch elm disease(DED) (Tainter & Baker, 1996). Since 1978, a moreaggressive fungus race, NAN (

Ophiostoma novo-ulmi

Brasier) has been responsible for a new epidemic, kill-ing thousands of elms in Spain (Muñoz & Rupérez,1980; Cadahía, 1983). In response to this disaster, theSpanish Forest Administration, in collaboration with

the Forestry School of the Universidad Politécnica deMadrid, initiated a breeding programme for the con-trol of DED in 1986 (Solla

et al

., 2000). The majorobjective of this programme was to obtain elms thatare resistant to this new race of fungal pathogen; how-ever, problems relating to seed abortion introducedsome difficulties to the breeding programme.

Ulmus minor

has hermaphroditic flowers. The gyn-oecium is normally formed by two carpels, though onlyone forms an ovule. Dichogamy is not marked and var-ies with climatic conditions. In most individuals, thestigma appears 2 or 3 days before anthesis. Otherindividuals, however, are slightly protandrous. Anthe-sis takes place in Central Spain during the secondfortnight of February and the first fortnight of March,varying with genotype and years. Pollination is ane-mophilous. After pollination, the fruit enlarges andforms a photosynthesizing samara. Maturation takesplace approximately 45–50 days after pollination. Thespecies is well known for the presence of empty sama-ras. In England, Stokes (1812), on describing

Ulmussurculosa

(considered

Ulmus minor

var.

vulgaris

according to Richens, 1983) indicated that its seeds

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rarely if ever ripen. This observation was later con-firmed by Ley (1910). Also in Spain it is well knownthat

Ulmus minor

produces a great percentage ofempty samara (Laguna, 1883; Catalán, 1985). Leliveld(1935) identified this problem in

Ulmus

¥

hollandica

,attributing it to the absence of fertilization. Partheno-carpy is also present in

Ulmus minor

(López-Almansa& Gil, 2003). As far as we know, no other species of thegenus has been previously cited as parthenocarpic(Schopmeyer, 1974), although we have observed it alsoin

U. glabra

Huds. and

U. laevis

Pall.The presence of empty fruits could be partially

related to the population structure and the lack ofcross pollination in the countryside (López-Almansa &Gil, 2003). However, during the 1999 cross-pollinationcampaign, it was also noted that seeds were aborted ina number of individual trees. No fertile seeds wereproduced, though shrunken, aborted seeds were visi-ble within the samara. In 2001 and 2002, we observedseed production in 40 randomly selected elms in ourclonal bank, and found that about 25% of the trees didnot produce any seed (López-Almansa, Pannell & Gil,2003), mainly due to seed abortion. In a natural standstudied in 2002, generalized seed abortion was evengreater, and it affected up to 76% of the 71 studiedtrees (López-Almansa

et al

., 2003). This further indi-cates that seed abortion is an extended problem inSpanish elms, and this has important evolutionaryconsequences.

In the course of our investigation, we realized thatthe embryological data on elm are very scanty. Fur-thermore, most of the current information is limited tothe early stages of embryo development (Shattuck,1905; Leliveld, 1935; Walker, 1938; Ekdahl, 1941;Crété, Guignard & Mestre, 1966; Guignard & Mestre,1967). Little is known concerning the maturation pro-cess of the embryo or endosperm development. Thepresent study was therefore undertaken (1) to detailthe structural pattern of embryo development and (2)to investigate the structural basis of embryo abortion.The study provides scientific knowledge that is essen-tial for the elm breeding programme.

MATERIAL AND METHODS

A

BORTION

AND

WATER

CONTENT

DETERMINATION

Developing seeds from three different field elms werecollected weekly at Centro de Mejora Genética For-estal de Puerta de Hierro in Madrid, central Spain, inMarch–April 2001. Maternal trees were clonal copiesof elms located in central (M-SF1), eastern (V-JR1)and northern (P-VV1) Spain. The first two clones, M-SF1 and V-JR1, were known for their abortion per-centages close to 100%; seed abortion was relativelyrare for the P-VV1 clone. M-SF1 morphologically

resembles

Ulmus minor

var.

vulgaris

, known in Brit-ain for not producing seeds (Ley, 1910; Richens, 1983).

Seed development was followed macroscopically,and seed abortion estimated using 50 seeds for eachgenotype and for each collection date. Those seeds inwhich shrinkage or chalazal necrosis had begun wereconsidered as aborted seeds. Average seed weight andwater content were determined using 30 seeds foreach genotype and date, after maintaining the seedsat 80

∞

C for 24 h.The study was repeated in March–April 2002,

adding five complementary trees (CC-VG2, CR-PB1,M-DV2, M-IN5, M-TC6), of which CC-VG2, M-IN5 andM-TC6 were known for aborting their seeds. Sampleswere collected weekly.

H

ISTOLOGICAL

PREPARATIONS

Approximately 20–30 developing seeds were collectedfrom each of three studied individuals (M-SF1, V-JR1,P-VV1) every 3–4 days from March to April during theyears 2000 and 2001. Seeds were fixed in 1.6%paraformaldehyde and 2.5% glutaraldehyde in 50 m

M

phosphate buffer, pH 6.8, for 24 h at 4

∞

C. After fixa-tion, samples were dehydrated in methyl cellosolve,followed by two changes of absolute ethanol, andembedded in Technovit 7100 (Yeung, 1999). Serial 3-

m

m sections were cut with glass knives on a Reichert-Jung 2040 Autocut rotary microtome. The sectionswere stained with the Periodic acid Schiff ’s (PAS)reaction for total insoluble carbohydrates, and coun-terstained with amido black 10B for proteins or tolu-idine blue O for general histological organization(Yeung, 1984). The sections were examined and pho-tographed using a Kodak colour print film, ASA 200,using a Leica Aristoplan and an Olympus BX50 lightmicroscope.

RESULTS

M

ACROSCOPIC

FEATURES

OF

SEED

DEVELOPMENT

The percentages of aborted seeds in 2001 and 2002 areshown in Figure 1. During the first stages of develop-ment, abortion could not be determined because seedswere too small.

In 2001, abortion was quite rare in P-VV1, and itnever reached over 6%. Germination was 100% inP-VV1. M-SF1 seed development appeared normalduring early development, and the abortion level wasrelatively low. However, during the sixth week afterfertilization, coincident with the seed desiccation pro-cess, abortion levels increased, reaching 98% on 16April. Seed abortion began early in V-JR1. A 40% seedabortion was noted 2 weeks after fertilization, on 22March. By the fifth week after fertilization, on 9 April,all seeds were dead.

ABORTIVE SEEDS IN

ULMUS MINOR

457



2004,

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, 455–467

The general pattern of development and the abor-tion process in these three individuals were very sim-ilar in 2002. CC-VG2, M-IN5 and M-TC6 showed apattern similar to V-JR1. CR-PB1 and M-DV2 behavedsimilar to P-VV1.

S

EED

WEIGHT

AND

WATER

CONTENT

The average fresh weight of the seeds is shown inFigure 2. In 2001, during the first 3 weeks after fertil-ization, which took place on about 8 March, bothM-SF1 and P-VV1 showed similar seed weights. In thefollowing week, the fresh weight of P-VV1 seedsincreased four-fold. By the fifth week, the maximumfresh weight was attained, with an average weight of13.8 mg seed

-

1

. At this time, the general desiccationprocess began and, within a week, the fresh weightdecreased by more than two-fold, resulting in a weightof only 6.1 mg seed

-

1

at the time of complete seed des-iccation. M-SF1 embryos often failed to expand. Themaximum fresh weight (5.59 mg seed

-

1

) was obtainedby the fourth week after fertilization. Desiccation pro-cesses started early, coinciding with the beginning ofthe seed abortion. About 6 weeks after fertilization, on19 April, when most of the seeds had aborted, theweight was only 0.88 mg seed

-

1

. It was not possible toobtain data for the V-JR1 genotype 3 weeks after fer-

tilization as seeds had begun to abort. In 2002, thechanges in the fresh weight patterns were similar tothat in 2001 for all three genotypes.

Changes in the dry weight of seeds are shown inFigure 3. In 2001, the three individuals maintainedsimilar weights until 29 March. Subsequently, P-VV1showed a rapid increase, but M-SF1 showed only asmall increase, and dry weight gained levelled offwhen abortion progressed. Because V-JR1 seedsaborted early, dry weight gain was not assessed3 weeks after fertilization. In 2002, the patterns of dryweight changes were similar to 2001.

S

TRUCTURAL

PATTERN

OF

EMBRYO

DEVELOPMENT

The control genotype (P-VV1)

Shortly after fertilization, the embryo begins todevelop (Fig. 4). Approximately 7 days after fertiliza-tion, the embryo reaches the early globular stage ofdevelopment (Fig. 5). The cells of the embryo properhave dense cytoplasm. The suspensor attachesdirectly to the inner surface of the inner integument(Fig. 5). The cells of the suspensor are more vacuolatedas compared with the cells of the embryo proper. Theendosperm expands rapidly after fertilization at theexpense of the nucellus tissue. Endosperm develop-

Figure 1.

Percentage of aborted seeds on three genotypesof European elm during late winter and early spring 2001and on eight genotypes of European elm during late winterand early spring 2002. Early abortives: M-TC6 (

�

, M-IN5(

▲

), CC-VG2 (

�

), V-JR (

�

); late abortive: M-SF1 (

¥

); non-abortive: P-VV1 (

�

), M-DV2 (

�

), CR-PB1 (

�

).

2002

2001100

80

60

40

20

0

2 3 4 5 6

100

80

60

40

20

0

1 2 3 4 5 6

Abo

rted

see

d (%

)

Weeks after fertilization

Figure 2.

Average fresh weight (mg) of developing seedson three genotypes of European elm during late winter andearly spring 2001 and on eight genotypes of European elmduring late winter and early spring 2002. Early abortives:M-TC6 (

�

), M-IN5 (

▲

), CC-VG2 (

�

), V-JR1 (

�

); late abor-tive: M-SF1 (

¥


�

), M-DV2 (

�

),CR-PB1 (

�

).

2002

2001

16

14

12

10

0

2

4

6

8

16

14

12

10

0

2

4

6

8

1 2 3 4 5 6

1 2 3 4 5 6

Weeks after fertilizationF

resh

wei

ght (

mg/

seed

)

458


ET AL

.



2004,

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, 455–467

ment conforms to the nuclear-type, in which divisionof the primary endosperm nucleus is not accompaniedby cytokinesis and results in the formation of freenuclei in the endosperm cavity. Cellularization occurslater, to form a partly cellularized endosperm. At theglobular stage, numerous nuclei line the endospermcavity and the developing embryo. The globularembryo continues to grow in size. Histodifferentiationbegins with the formation of a distinct protodermlayer (Fig. 6).

Approximately 10 days after fertilization, theembryo transforms into a heart-shaped embryo withthe formation of cotyledons (Fig. 7). Apical meristemsare clearly discernible. In the embryo, the procam-bium also begins to form, which traverses between theshoot and root apical meristems. Cotyledons expandrapidly by vacuolation and gradually extend towards

the chalazal end of the seed. Simultaneously, cellular-ization begins with cellular endosperm forming nearthe developing embryo and next to the developing seedcoat. Cellular endosperm cells are formed in a centri-petal manner. A greater number of cellular endospermcells are present around the developing embryo(Fig. 7). The cellular endosperm comprises highly vac-uolated cells with thin walls. Liquid endospermremains at the centre of the endosperm cavity. At thechalazal end, the endosperm is in close contact withthe nucellus, reaching the proximity of the hypostasis,and develops into a densely cytoplasmic coenocytichaustorium with many nuclei (Fig. 8).

About 15 days after fertilization, the cotyledons con-tinue to expand with a well-defined embryo axis(Fig. 10). Both the root and the shoot apical meristemsare well defined and the meristems are separated bythe procambium. Cellular endosperm occupies most ofthe endosperm cavity, although sometimes a ‘hole’without cells is present near the chalazal end. Theendosperm cells adjacent to the embryo contain PAS-positive staining material (Fig. 9). As the embryoexpands in size, the cellular endosperm cells adjacentto the embryo are gradually crushed and the contentsare absorbed by the expanding embryo. Theendosperm cells lining the endosperm cavity aresmaller in size than those located at the centralregion.

The embryo grows rapidly and it occupies most ofthe endosperm cavity. The endosperm is reduced to theparietal layer, with a width of one or two cells(Fig. 11), except at the chalazal end, where multilay-ers of endosperm cells are still present and have notyet degenerated. The endosperm haustorium is nolonger present. At this time, the tissue pattern of theembryo is clearly discernible. The procambium is alsowell developed within the cotyledons (Fig. 12). Allembryo cells at this stage are vacuolated. A smallnumber of tiny starch grains are present within cells.

By 28–30 days after fertilization, storage productsbegin to accumulate within embryo cells (Figs 13, 14).Initially, starch is more abundant within the cells(Fig. 13). Large vacuoles gradually reduce in size andsmaller vacuoles appear within the cytoplasm(Fig. 14). Proteins begin to accumulate within thesesmall vacuoles. The amount of starch gradually

Figure 3.

Average dry weight (mg) of developing seeds onthree genotypes of European elm during late winter andearly spring 2001 and on eight genotypes of European elmduring late winter and early spring 2002. Early abortives:M-TC6 (

�

), M-IN5 (

▲

), CC-VG2 (

�

), V-JR1 (

�

); late abor-tive: M-SF1 (

¥


�

), M-DV2 (

�

),CR-PB1 (

�

).

2002

2001

0

1

2

3

4

5

6

7

0

1

2

3

4

5

6

7

1 2 3 4 5 6

1 2 3 4 5 6

Dry

wei

ght (

mg/

seed

)

Weeks after fertilization

Figures 4–9.

Light micrographs of P-VV1 (normal genotype). Fig. 4. Section of ovule showing the first stage of embryodevelopment. Nucellus is well developed (asterisk), and has more cytoplasmic cells at the chalazal end. Fig. 5. Earlyglobular embryo, which is densely cytoplasmic. Suspensor (arrow) cells are more vacuolated. Nuclear endosperm lines theembryo. Starch is present in the nucellus (arrowhead). Fig. 6. Late globular embryo showing a distinct protoderm layer(arrow). Nuclear endosperm still lines the embryo. Fig. 7. A heart-stage embryo with expanding cotyledons. Apicalmeristems are visible, and endosperm close to the embryo is cellularizing. Fig. 8. Chalazal end showing a well-developedhaustorium (arrow) and a polyphenolic-rich hypostasis (asterisk). Fig. 9. Cellular endosperm showing PAS-positive stain-ing material (arrow) accumulation between cells. All scale bars = 30

m

m.

ABORTIVE SEEDS IN

ULMUS MINOR

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2004,

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4

5

7

8

96

460


ET AL

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Botanical Journal of the Linnean Society, 2004, 145, 455–467

reduces in the cytoplasm of the cells. At the time whenthe embryo matures (Fig. 15), little starch is presentwithin the cotyledons and the embryo axis. The cyto-plasm of the embryo cells is filled with protein bodies.Although storage lipids have not been stained for, thevesicular nature of the cytoplasm indicates the pres-ence of storage lipids within the embryo. Theendosperm is reduced to just one to two layers nearthe seed coat and small protein bodies are also presentwithin the cytoplasm of the cells.

A large amount of nucellus is present enveloping theembryo sac at the time of fertilization (Fig. 4). At thechalazal end, nucellar cells remain small and have adenser cytoplasm. Starch is present within the nucel-lar cells during the early stages of embryo develop-ment. More starch is present at the micropylar end ofthe nucellus (Fig. 5). At the region where the nucellusjoins with the integumentary cells, phenolic sub-stances are very abundant within cells, forming ahypostasis (Fig. 8). The nucellus is gradually absorbedby the expanding endosperm and it completely disap-pears (Fig. 13) as the embryo matures, leaving a thinlayer of cell wall material.

The seed coat consists initially of approximatelyeight cell layers except in the region of the vascularbundles where more cell layers are present. The cellsof the outermost layer as well as the inner cell layernear the chalazal area accumulate a large amount ofphenolic substances (Figs 10, 15). Vascular tissues arewell developed within the seed coat and they termi-nate near the chalazal end. At the time of maturation,the middle layers of the seed coat become greatly com-pressed by the expanding embryo. In a fully matureseed, only the outermost and the innermost cell layersremain (Fig. 15).

The late abortive genotype (M-SF1)The first stages of seed development are similar to thecontrol genotype (P-VV1) (Fig. 16). At 12–14 daysafter fertilization, cellularization begins within theendosperm and embryos are at the globular stage. Cot-yledons expand laterally, resulting in the formation ofa heart-shaped embryo. Cellular endosperm cells are

present enveloping the developing embryo and gradu-ally fill the entire endosperm cavity.

Abnormalities begin to appear about 20 days afterfertilization. Embryo development appears to bedelayed (Fig. 17). PAS-positive material begins toaccumulate in the central endosperm cells. These cellssubsequently degenerate, releasing the PAS-positivematerial (Fig. 18). The cellular endosperm cells nearthe seed coat differ from those near the developingembryo. These parietal endosperm cells are smaller insize and are 1–2 layers thick, except towards the cha-lazal end, where 2–5 layers of endosperm cells can befound. However, in some developing seeds the parietalendosperm is more developed; 4–5 layers ofendosperm cells line the entire endosperm cavity(Fig. 19). Later, the vacuoles that initially occupy mostof these endosperm cells divide and stain moreintensely with a protein stain (Fig. 20). Frequently, asmall amount of starch is also present within theircytoplasm.

During the following 10 days, some embryos con-tinue to expand and eventually occupy most of theendosperm cavity (Fig. 21). Nevertheless, the matura-tion process is delayed, and the pattern of storage pro-tein deposition is also abnormal as more storageprotein bodies are formed in the embryo cells near theseed coat. In general, a majority of the embryos appearsmaller as they fail to expand and embryo cells remainvacuolated (Fig. 22). The absence of storage productformation in many embryos results in the embryo col-lapsing as the seed dries. Furthermore, some of theembryos never reach the heart stage and remain as ahighly cytoplasmic globular embryo (Fig. 23). Theseembryos die when the desiccation process begins.

The early abortive genotype (V-JR1)The first steps of development appear to be normal,and embryo development continues through to theglobular stage. The endosperm cellularizes early,between 5 and 10 days after fertilization, when theembryo is still at the globular stage (Fig. 24). Thehaustorium develops well. Soon after cellularizationof the endosperm, a large amount of PAS-positive

Figures 10–15. Light micrographs of P-VV1 (normal genotype). Fig. 10. A cotyledon-stage embryo with expanding coty-ledons. The apical meristems are clearly discerned (arrowhead). Fig. 11. Section showing the endosperm, the nucellus (nc)and the seed coat (sc). Cellular endosperm produces PAS-positive staining material (asterisk) in the most internal layer,while the parietal endosperm (pe) close to the nucellus begins to accumulate proteins. Fig. 12. Procambium (asterisk) isclearly distinct as cotyledons mature and the cotyledon cells are vacuolated at this time. Fig. 13. Section showing a partof the embryo, the parietal endosperm (pe) and the seed coat (sc). Polyphenolics accumulate in the outer layer of the seedcoat (arrow). Nucellus has disappeared. Fig. 14. Embryo begins to accumulate storage products. Starch begins to appearfirst (arrowheads). Soon, vacuoles reduce in size and begin to accumulate proteins (arrow). Fig. 15. Mature embryo. Starch,though present, is not very frequent. A large number of protein bodies are present (arrows) within cells. The parietalendosperm layer also reacts positively to protein stain; however, protein bodies are not as abundant. The seed coat becomeshighly compressed. All scale bars = 30 mm.

ABORTIVE SEEDS IN ULMUS MINOR 461

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462 J. C. LÓPEZ-ALMANSA ET AL.


material begins to accumulate within the central cel-lular endosperm cells near the developing embryo(Fig. 25). Embryo development appears to be retardedor arrested with the formation and accumulation ofcarbohydrate materials in the cellular endospermcells. Sometimes embryos are able to form the cotyle-dons, but deposition of storage products is not present(Fig. 26). Approximately 25 days after fertilization,the degeneration process is advanced (Fig. 27). Theinteguments have degenerated in the chalazal regionand the nucellus has completely disappeared. Theparietal endosperm is 2–3 cells thick, and it accumu-lates a great number of protein bodies (Fig. 28). Amassive amount of carbohydrates, greater than theother two genotypes, as judged by the intensity ofcolour development towards the PAS reaction, ispresent in the degenerating endosperm. When desic-cation begins, inward tensions make the seed coatwrinkle and collapse into the endosperm cavity(Fig. 29). Shortly thereafter, degeneration is complete,and only some amorphous, black and shrunken tissuesremain within the samara.

DISCUSSION

The first stages of seed development in P-VV1 aresimilar to those described by Leliveld (1935) inUlmus ¥ hollandica and to those described by Corre-doira, Vietez & Ballester (2002) in Ulmus glabra. Inthis study, we have extended the description throughto seed maturation in Ulmus minor. Similar to otherflowering plants, the embryo progresses throughdefined morphological stages leading to developmen-tal arrest as the seed matures. Storage products beginto accumulate after the cotyledon stage of embryodevelopment. At the time of maturation, the embryocells are filled with storage products. This is reflectedby the continuous increase in the dry weight of theseeds.

Initial development of the endosperm in genotype P-VV1 resembles the nuclear type of endosperm devel-opment (Vijayaraghavan & Prabhakar, 1984), inwhich division of the primary endosperm nucleus isnot accompanied by cytokinesis. Cellularization of theendosperm occurs at the heart stage of embryo devel-

opment. When compared with the more typical exam-ples of the nuclear type endosperm such as in thePhaseolus species (Yeung & Cavey, 1988), a moreextensive cellularization of the endosperm is notedprior to being crushed by the expanding embryo. How-ever, as the seed matures, only a thin parietal layer ofcellular endosperm remains near the seed coat. Fur-thermore, during the course of cellular endospermdevelopment, a small amount of PAS-positive materialis synthesized by the endosperm cells located near thedeveloping embryo. This material never accumulateswithin seeds and it disappears as the embryo matures.The general pattern of endosperm development in P-VV1 is similar to that described by Chernik (1983) inUlmus laevis. The presence of a chalazal haustoriumhas been previously reported in U. ¥ hollandica (Leliv-eld, 1935) and in other species of the family Ulmaceaes.l. (Dottori, 1994).

It is clear from this study that seed abortion in theabortive genotypes is not due to the absence of fertil-ization, as no apomixis has been found in our matingstudies (López-Almansa, 2002). In both abortive gen-otypes, embryo development progresses normallyuntil the globular stage. Subsequent to this stage,embryo development is delayed or arrested. Evenwhen the embryo is able to develop further, abnormal-ities such as a failure to accumulate storage productsare often observed. The marked difference in embryodevelopment between the control and the abortivegenotypes is most likely caused by differencesobserved in endosperm development. Abnormality ofendosperm development is one of the main causes ofembryo abortion in flowering plants (Brink & Cooper,1947). The structural pattern of endosperm develop-ment in the abortive cell lines is abnormal as judgedby the early cellularization and the massive synthesisof PAS-positive material, especially in V-JR1.Although the endosperm is generally regarded as animportant nutrient source for the developing embryo,as judged from the precocious development of theendosperm after fertilization (Yeung & Clutter, 1978)and its negative water potential (Yeung & Brown,1982), the endosperm may in fact compete with thedeveloping embryo for nutrients. The altered patternof endosperm development in the two abortive

Figures 16–23. Light micrographs of M-SF1 (late abortive genotype). Fig. 16. Globular embryo with nuclear endosperm.Scale bar = 30 mm. Fig. 17. Delayed embryo surrounded by PAS-positive carbohydrate material (asterisk). Scalebar = 30 mm. Fig. 18. PAS-positive carbohydrate material (asterisk) released by cellular endosperm. Scale bar = 30 mm.Fig. 19. Parietal endosperm (pe), with a greater cell proliferation as compared with the control. Scale bar = 30 mm. Fig. 20.Parietal endosperm (pe) and seed coat (sc). The parietal endosperm shows an extensive accumulation of protein bodies.Scale bar = 30 mm. Fig. 21. Maturing embryo showing an abnormally high protein deposition in the embryo cells near theseed coat (arrowheads). Scale bar = 200 mm. Fig. 22. Vacuolated embryo with abnormalities in the cotyledons. Scalebar = 100 mm. Fig. 23. Small globular, cytoplasm-rich embryo (arrowhead) surrounded by PAS-positive carbohydratematerial. Scale bar = 100 mm.



2319

18

17

1620

22

21



genotypes of elm may have further shifted the equi-librium of nutrient competition in favour of theendosperm. The synthesis and accumulation of PAS-positive material in the central endosperm cells, andthe accumulation of storage protein bodies in the pari-etal endosperm layers, could be a direct result of anincreased ability of the endosperm to acquire nutri-ents from the maternal tissue, i.e. the seed coat. Thiscould result in the failure to translocate nutrients tothe developing embryo, leading to embryo abortion.The slow dry weight gain as shown in Figure 3 sup-ports the above hypothesis.

The synthesis and extensive accumulation of PAS-positive material in the abortive genotypes may have anegative effect on embryo growth. This material isgluey in nature; during seed dissection such a sub-stance is found in the endosperm of the abortive celllines. This substance resembles a type of mucilage.Because mucilage is known for its water-binding capa-bility (Mauseth, 1988), a large amount of mucilagepresent within the endosperm could reduce theamount of water available for embryo expansion. Thismay explain why embryo development is retarded,especially for the V-JR1 line in which massiveamounts of mucilage are present and some embryosare arrested at the globular stage.

The production and accumulation of PAS-positivematerial in the endosperm will also alter the qualityand quantity of nutrients available to the developingembryo. The synthesis of this material and the subse-quent synthesis and accumulation of storage proteinsin the parietal endosperm cells could also shield nutri-ents from reaching the developing embryo. Althoughsome embryos could develop further, a reduction innutrient availability may have resulted in a lesseramount of storage products accumulated within theembryo. The lack of storage products within cellsmakes these embryos more susceptible to damage dur-ing maturation drying. As a result, the embryos do notsurvive and a majority of the seeds from M-SF1 and V-JR1 genotypes abort (Fig. 1).

Developmental events are highly regulated. Theevents observed during embryo development, such asembryo morphogenesis and the pattern of storageproduct deposition observed during seed development,

are programmed processes. This is most likely inresponse to the stable embryonic environment enclos-ing a developing embryo (Yeung, 1995; Yeung, Meinke& Nickle, 2001). In a preliminary study, embryos fromthe abortive genotypes could be rescued and culturedusing the in vitro embryo culture method, although noplantlets were obtained (López-Almansa, 2002). Thisindicates that embryo development could be normal,taking into account the difficulties of embryo culturein elm (Corredoira et al., 2002). In the abortive celllines, embryo abortion could be a result of a mis-coor-dination between embryo and endosperm develop-ment. Irregularities in timing between embryo andendosperm development had already been noted inelm by Leliveld (1935), although she considered themof no further importance. In Arabidopsis, it has beenshown that mutations can confer an autonomousendosperm development in the absence of fertilization(Vinkenoog & Scott, 2001). In the abortive genotypesof elm, epigenetic or genetic changes could lead to pre-cocious development of the endosperm, disrupting theco-ordinated pattern of development between theembryo and the endosperm.

The endosperm balance number (EBN) hypothesissuggests that a ratio of two maternal (m) to one pater-nal (p) of effective ploidy levels in the endosperm isneeded for the correct development of the seed in spe-cies with triploid endosperm (Johnston et al., 1980). Inmaize, Lin (1984) demonstrated that the critical factorfor normal endosperm development is a 2m : 1pgenomic ratio rather than a specific ploidy. Similarconclusions have been drawn using Arabidopsisthaliana (Scott et al., 1998), in which crosses 6x(maternal) ¥ 2x (paternal) produced an abnormal seeddevelopment characterized by embryo arrest and earlyendosperm cellularization in a manner very similar tothat we observed in Ulmus minor. This suggests thatdifferences in the ploidy levels could account for gen-eralized seed abortion in elm. However, the abundanceof well-developed seeds in controlled crosses using theabortive lines as the paternal source, and the lack ofseed when crossing only abortive genotypes (López-Almansa, 2002), is different from the behaviour shownby Arabidopsis, suggesting that no ploidy differencesare present. A possible explanation could be the

Figures 24–29. Light micrographs of V-JR1 (early abortive genotype). Fig. 24. Late globular embryo surrounded bycellular endosperm. Cellular endosperm is advanced as compared with control. Scale bar = 30 mm. Fig. 25. Delayed embryoat transition stage, surrounded by carbohydrate-rich endosperm. Scale bar = 30 mm. Fig. 26. Embryo in the cotyledon stage.Endosperm mucilage is very abundant (asterisk). The embryo cells remain vacuolated. Scale bar = 30 mm. Fig. 27.Advanced degeneration of the seed. The embryo (em) does not accumulate storage products. The endosperm mucilage hasa very intense PAS reaction (asterisk). Chalazal integuments are necrotic. Scale bar = 60 mm. Fig. 28. Parietal endosperm(pe) with a great accumulation of protein bodies. PAS reaction is very intense in the mucilage (asterisk). Scale bar = 30 mm.Fig. 29. Seed in the last stage of development, after desiccation begins, showing the seed coat collapsing into the endospermcavity due to inward tension. Scale bar = 60 mm.



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presence of differences in ploidy levels not just in thesporophyte but in the female gametophyte, as a resultof a failure during sporogenesis, although at presentthis is only a working hypothesis.

Environmental conditions could play a minor role inthe aborting process, as revealed by the slight differ-ences in M-SF1 behaviour in 2001 and 2002. However,it seems clear that abortion is a constant pattern, andthus four abortive individuals monitored between1999 and 2002 aborted their seed every year (López-Almansa, 2002). Those trees with a generalizedabortion cannot reproduce through seed, and theirreproductive capability is limited to pollen. As aconsequence, in those places where abortive and non-abortive trees live together, a functional subandrodio-ecy might be present (López-Almansa et al., 2003).However, this interpretation is not clear owing to thecomplex taxonomic classification of Ulmus minor s.l.(López-Almansa, 2004).

Further studies including ploidy analysis of thesporophyte and gametophyte, and cross-pollinationbetween various genotypes, are needed for testingthese possibilities and may provide additional insightsinto embryo abortion in elms. Presence of generalizedabortion in some elm genotypes limits their reproduc-tion capability to the male function and could haveimportant evolutionary consequences. The results wehave obtained open a line of enquiry that can clarifythe genetic basis of the reproductive behaviour ofEuropean field elm and its evolutionary significance.

ACKNOWLEDGEMENTS

This research was partially funded by the SpanishDirección General de Conservación de la Naturaleza,Ministerio de Medio Ambiente. J.C.L.A. was supportedby an FPU scholarship and a complementary grantfrom MECD (Ministerio de Educación, Cultura yDeporte). This research was also supported by anNSERC (Canada) research grant to E.C.Y. NataliaLoukanina provided a translation of the paper by V. V.Chernik. José Gutiérrez-Muñoz and an anonymousreferee suggested modifications to an earlier version ofthe manuscript.

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Abortive seed development in Ulmus minor (Ulmaceae)

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