sucrose synthase localizati n during lnitiation of seed...

Plant Physiol. (1 995) 109: 1285-1 293

Sucrose Synthase Localizati n during lnitiation of Seed Development and Trichome Differentiation in Cotton Ovules’

Kurt D. Nolte, Donald 1. Hendrix, John W. Radin, and Karen E. Koch*

Horticultural Sciences Department, 11 51 Fifield Hall, University of Florida, Gainesville, Florida 3261 1 (K. D. N ., K. E. K.); Western Cotton Researc h Laboratory, U n ited States Department of Agricu Itu re-Agricu ltu ral

Research Service, 41 35 East Broadway, Phoenix, Arizona 85040 (D.L.H.); and National Program Staff, United States Department of Agriculture-Agricultura1 Research Service, Building 005, Beltsville Agricultural Research

Center West, Beltsville, Maryland 20705-2350 (J.W.R.)

Sucrose synthase in cotton (Gossypium hirsutum L.) ovules was immunolocalized to clarify the relationship between this enzyme and (a) sucrose import/utilization during initiation of seed development, (b) trichome differentiation, and (c) cell-wall biosynthesis in these rapidly elongating “fibers.” Analyses focused on the period immediately before and after trichome initiation (at pollination). Interna1 tissues most heavily immunolabeled were the developing nucellus, adjacent integument (inner surface), and the vascular region. Little sucrose synthase was associated with the outermost epidermis on the day preceding pollination. However, 1 d later, immunolabel appeared specifically in those epidermal cells at the earliest visible phase of trichome differentiation. l h e day following pollination, these cells had elongated 3- to 5-fold and showed a further enhancement of sucrose synthase immunolabel. Levels of sucrose synthase mRNA also increased during this period, regardless of whether pollination per se had occurred. Timing of onset for the cell-specific localization of sucrose synthase in young seeds and trichome initials indicates a close association between this enzyme and sucrose import at a cellular level, as well as a potentially integral role in cell-wall biosynthesis.

Cotton ovules provide an experimental system with dual advantages. First, specific aspects of Suc synthase localization during early seed development can be examined in a well-defined, representative dicotyledonous species. The structural features of the growing ovules can be readily distinguished (diagrammed in Fig. 11, and these corre- spond to specific phases of development. As a result, it has been possible to correlate changes in metabolite levels and enzymatic activities of partially dissected seeds with structural alterations during their formation (Jaquet et al., 1982; Hendrix, 1990). Imported sugars also support both respi- ration and biosynthesis in this system, with the latter including constituents for growth of the embryo, endosperm, and nucellus and extensive elongation of epidermal hairs

This research was supported by a grant from the National Science Foundation (Cellular Biochemistry) and by a cooperative agreement between the U.S. Department of Agriculture and the Florida Agricultura1 Experiment Station (Journal Series No.

* Corresponding author; e-mail kek8gnv.ifas.ufl.edu; fax R-04806).

1-904 -392- 6479.

(Trelease et al., 1986). Although carbon for these functions originates from SUC entering the ovule (Basra and Malik, 1984), the processes involved after Suc exit from the phloem remain unclear.

Second, fibers of the cotton seed exhibit a remarkable capacity for cell-wall biosynthesis and thus facilitate the study of related processes. Each of these fibers i s a single trichome cell that emerges from the outermost layer of epidermis on the seed. Trichomes within a given boll share a precise synchrony of development and homogeneity during growth (Ryser, 1985). In addition, the cotton fiber has proved to be an excellent model system for the study of cell growth and cellulose biosynthesis (Meinert and Delmer, 1977; Delmer, 1987; Timpa and Triplett, 1993; Delmer and Amor, 1995). Trichome cell-wall production represents a substantial portion of overall seed growth (Hendrix, 1990, and refs. therein). In fiber cells themselves, final dry weight can be attributed to 95% cellulose (Timpa and Triplett, 1993). Earlier in fiber development, when elongation is most rapid, cellulose content is closer to 35 to 50%, with pectin and hemicellulose making up much of the difference (Meinert and Delmer, 1977). UDP-Glc appears to be the preferred substrate for cellulose biosynthesis (Carpita and Delmer, 1981; Delmer, 1987) and can also be readily con- verted to other UDP sugars for production of hemicellulo- ses and pectin (Amino et al., 1985). The UDP-Glc could arise from Suc and UDP via Suc synthase (EC 2.4.1.13), and/or from Glc-1-P and UTP via UDP-Glc pyrophosphorylase (EC 2.7.7.9). Although activity of the latter is often greater than that of Suc synthase (Wafler and Meier, 1994), the direction of the reaction may be less likely to produce UDP-Glc (Delmer and Amor, 1995). Suc synthase could provide not only a direct pathway of C transfer from newly imported Suc to UDP-Glc, but also one theoretically less costly in overall ATP use. Both Suc synthase and invertase activities have previously been reported in cotton ovules (Beasley et al., 1974). In addition, Hendrix (1990) has found Suc synthase to be especially active in extracts of young ovules during most rapid trichome elongation. Delmer et al. (1995) have even proposed that one form of this enzyme may be physically associated with cellulose synthase. A cell-leve1 analysis of Suc synthase distribution was under- taken in the present study to further address the extent of its relationship to expanding fibers.

1286 Nolte et al. Plant Physiol. Vol. 109, 19!15

vascular bundle /

chalazal Ci

nucellus

inner integumen

outer integumen ulus

micropyle ’

Figure 1. Longitudinal diagram of a cotton ovule showing location and morphology of tissues at flower openinghnthesis.

The following research documents a specific association between SUC synthase and precise changes in both timing and site of rapid elongation/cell-wall biosynthesis. In newly initiated cotton trichomes, the appearance of Suc synthase marked one of the first signs of onset of this developmental program. Additionally, the clear cytoplasmic localization was accompanied by prominent immunolabel near the cell surface and in elongating regions of these cells. Both observations are consistent with the hypothesis that SUC synthase has an integral role in formation of cell-wall constituents, catalyzing the first step in their synthesis by directly transferring C from Suc to UDP-Glc. The additional presence of Suc synthase in the nucellus and innermost surface of the enveloping integument also sup- ports its probable contribution to elevated respiratory and biosynthetic processes in these tissues.

MATERIALS A N D M E T H O D S

Plant Mater ial

Acala SJ-1 cotton plants (Gossypium hirsutum L. [obtained from the Agronomy Department, University of Florida, Gainesville]) were grown during the fall production season in 1992 (August to November; pollination occurred about September to November) in the field in 200 m x 1 m rows, spaced 1 m apart. Commercial cultural practices were em- ployed as described by Benedict et al. (1976). On the after- noon prior to flower opening (1-2 PM, Eastern Standard Time), 10 flower buds were harvested and 20 others were tagged. Ten of the latter were sampled the following day (flower opening/anthesis) and again 1 d later. Harvested buds/flowers were kept at 4°C during transport to the lab and were immediately dissected. Ovary walls were excised with a scalpel, and the ovule-filled loculus was quickly transferred to formalin acetic acid for preservation. For emasculation experiments, anthers were carefully excised

prior to flower opening/anthesis and flowers were bagged immediately to prevent insect pollination.

Immunolocalization

The immunogold silver-staining reactions were adalpted from the specifications outlined by Nolte and Koch (1993). For cotton ovules, tissues were sectioned (15 pm), affixed to slides, deparaffinized, rehydrated, and washed with PBS. Overnight incubation with 300 FL of 5% heat-inacti- vated normal goat serum (about 10 h) was followed by a 1-h treatment with an equal amount of 1:75 diluted Suc synthase polycl onal antisera. The concentrations used were based on the greatest possible dilutions that would still provide a strong positive signal for the protein. After three 10-min washes with PBS solution, slides were incubated for 1 h in a humid environment with a 1:200 dilution oí secondary antibody (goat anti-rabbit IgG linked to 5-nm col- loidal gold [Zymed Laboratories, Inc., South San Francisco, CAI). Polyclonal antibodies had been raised against a com- bination of Shl and Susl gene products extracted from maize kernels (W64A X 182E) 22 d after pollination. Slides were then washed three times for 10 min each with PBS buffer and three times for 5 min each with distilled water, incubated for 15 min with freshly prepared silver enhancement reagents, and washed with excess distilled water. Slides were counterstained and permanently mounted for microscopic evaluation.

R N A Extractioii and Northern Blotting

Cotton ovules were ground to a fine powder in a mortar and pestle with liquid N, and RNA was isolated essentially according to McCarty (1986). This method was modified for remova1 of especially high levels of phenolic com- pounds in cotton ovules by adding polyvinylpolypyrroli- done (20%, w/v) to the extraction medium and again to each of four subsequent phenol/chloroform rinses. Total RNA was quantified by UV spectroscopy at 260 nm. RNA samples were denatured in 2.2 M formaldehyde and frac- tionated on 1.0% agarose/2.2 M formaldehyde gels (Tho- mas, 1980), blotted onto a nylon membrane (Hybond-N, Amersham), and probed according to Church and Gilbert (1984). SUC synthase clones (cDNA for Shl and genomic for Susl) were obtained from L.C. Hannah (University oí Flor- ida, Gainesville, FL) and were radiolabeled by random primer extension. Blots were rinsed and placed on x-ray film at -80°C.

RESULTS

A clearly defined differential distribution of Suc synthase protein was evident during initiation of cotton seed and trichome development. In ovules harvested immediately prior to flower opening/anthesis, polyclonal anti- serum to SUC synthase specifically labeled cells in the nucellar tissue and in parenchyma adjoining the vascular bundle (Fig. 2, A and D). The latter included the entire area near the site of Suc unloading from phloem at the chalazal end of the ovule (Fig. 2, A and D). Immunogold stain was not readily detected in other ovule cells such as those of the

Sue Synthase Localization in Cotton Ovules 1287

D

B

E

H

K

\

F •'•*,',•

L

Figure 2. Light micrographs of cotton ovulessampled either 1 d before, the day of, or 1 d afterflower opening/anthesis showing areas of Suesynthase immunogold localization at low mag-nification (33X). A, D, G, and J, Longitudinal (A)and transverse (D) sections of cotton ovules be-fore flower opening/anthesis showing distribu-tion of immunolocalized Sue synthase withinnucellar tissue and cells associated with the vas-cular bundle. Sue synthase label was not de-tected in longitudinal (G) or transverse (J) sec-tions treated with preimmune serum (controls).B, E, H, and K, Longitudinal (B) and transverse(E) sections of cotton ovules on the day of floweropening/anthesis showing Sue synthase immu-noreactivity in newly forming epidermal hairsand cells of the nucellus and vascular bundle.Little or no immunogold label was detected inlongitudinal (H) or transverse (K) sections withpreimmune serum (controls). C, F, I, and L, Lon-gitudinal (C) and transverse (F) sections of cottonovules 1 d after flower opening/anthesis show-ing strong immunoreactivity of Sue synthase inepidermal hairs and nucellar and vascular tis-sues. Note the absence of immunolabel in lon-gitudinal (I) and transverse (L) sections with pre-immune serum (controls). Bar = 250 /nm.

1288 Nolte et al. Plant Physiol. Vol. 109, 19'35

epidermis, either of the two integuments, or the chalazal cap zone. Ovule development on the day of flower opening/anthesis and on the day immediately following this was highlighted by initiation of elongation in epidermal trichomes in the outer integument and the concomitant appearance of immunolocalized Suc synthase in these cells (Fig. 2, B, D, E, and F). Except for this change in labeling of specific epidermal cells, no other noticeable differences were detected in the pattern of immunolocalization among outer integuments as ovules progressed from pre- to postanthesis. However, during this same period, an accumula- tion of Suc synthase protein was evident in the cells of the innermost surface of the inner integument (Fig. 2, C and F). A distinct association between Suc synthase localization, hair development, and proximity to the vascular strand was also apparent in transverse sections of ovules 1 d postanthesis (Fig. 2F). Those trichomes at the chalazal end of the ovule elongated most rapidly and were also most proximal to the zone of phloem unloading.

Epidermal trichomes of cotton ovules were more closely examined before, during, and after flower opening/anthesis to investigate the distribution of Suc synthase within these rapidly growing cells (Figs. 3 and 4). Prior to flower opening (Fig. 4, A-C), most epidermal cells of the ovule (about 95%) had a well-defined nucleus, numerous small vacuoles, and showed little or no Suc synthase immunolabel (Figs. 3D and 4A). A few epidermal cells (about 5%) had distinctly larger central vacuoles and showed a slightly greater degree of Suc synthase immunolabeling (Fig. 4 0 .

Within 24 h, a large, spherical expansion above the ovu- lar surface was evident in each of a small portion of epidermal cells (Figs. 3, B and E, and 4, D-F). Trichome initials first appeared on the chalazal surface of the ovule (Fig. 3B), and others later became evident at the micropylar end (Fig. 3C). Within the epidermis, a heavy, black immunogold label was present in the cytoplasm of expanding trichomes and near the outermost surface of the enlarging region of these cells (Fig. 4, D and F). This peripheral localization is not inconsistent with a plasma membrane association for some of the SUC synthase in these cells (recently suggested by Delmer et al., 1995). Nondifferentiating cells and those underlying the epidermis showed no detectable immunoreactivity. Spherical expansion was followed by cell elongation on the day after flower opening/anthesis (Figs. 3, C and F, and 4, G-I). The pattern of immunostaining in these cells was essentially similar to that observed 24 h earlier, although the overall abundance of Suc synthase immunolabel was still greater.

Suc synthase message levels showed a similar change when examined in cotton ovules before, during, and after flower opening/anthesis of intact control (pollinated) versus emasculated (nonpollinated) cotton flowers (Fig. 5). Low levels of Suc synthase transcript were detected in ovules from emasculated and intact flowers prior to flower opening/anthesis; these levels increased sharply during flower opening/anthesis and increased still more thereaf- ter (Fig. 5). In addition, control flowers left intact (pollinated) and those that had been emasculated (nonpollinated) had similar levels of Suc synthase transcript. Trichome

initiation and growth were also similar in control and emasculated flowers during this developmental period (data not shown). Apparently, induction of Suc synthase and initial trichome development were both independent of pollination.

DI SCUSSION

Suc synthase in cotton ovules was immunolocalized to clarify the relationship between this enzyme and (a) Suc import/utilization during ovule development, (b) trichome initiation, and (c) cell-wall biosynthesis in these rapidly elongating cells. Although activity is known to be high in endosperm during grain fill (Chourey and Nelson, 1976), its significance and localization remain unclear during the early stages of development in ovules of both monocoty- ledonous and dicotyledonous plants (Xu et al., 1989; Hen- drix, 1990). In addition, the rapid expansion of newly initiated cotton trichomes gives this system a special advantage for an appraisal of Suc synthase relative to cell-wall biosynthesis. Previous researchers have suggested that Suc synthase may provide UDP-Glc for cell-wall formation based on biochemical studies (Carpita and Delmer, 1981; Basra and Malik, 1984; Delmer, 1987), mutant pheno- types (Chourey et al., 1991; B. McClintock, unpublished data), and transient expression systems (Maas et al., 1990). This hypothesis has been difficult to test but is addressed here in relation to highly localized synthesis of cell-wall constituents (versus storage products) (Wang et al., 1994).

An initially surprising addition to the trichome immunolabeling in the present study (discussed below) was the presence of Suc synthase in and near the nucellus of recently fertilized ovules (Figs. 2 and 3) . This localization is reminiscent of that observed later in maize endospl-rm (Chourey and Nelson, 1976; Chourey et al., 1991) or in developing bean cotyledons (Xu et al., 1989; Sung et al., 1994). Like these structures, the nucellus represents a predominant tissue mass in the ovule or seed at a given time, and provides a large, transient storage reserve for the riext stage in development (Jensen, 1965). It is positioned between the embryo sac and innermost integument, and con- tinues to grow, store, and supply nutrients until it is even- tually crushed and absorbed by the enlarging embryo (Jensen, 1965; Mansfield and Briarty, 1990). The ultimate nutritive role of this maternal tissue depends on its capacity to import assimilates and convert them to starch, protein, and lipid (Jensen, 1965; Mansfield and Briarty, 1990).

In this respect, the function of Suc synthase in the nucellus of a young cotton seed parallels that in developing cotyledons and endosperm during seed fill. Its action can supply UDP-Glc not only for biosynthesis of starch (Chourey and Nelson, 1976) and/or cell walls (Maas et al., 1990; Chourey et al., 1991), but also for efficient entry of carbon into the respiratory path (Xu et al., 1989, and refs. therein). In the nucellus, contributions by Suc synthase to starch storage (Jensen, 1965) and extensive cell-wall thiick- ening (Jensen, 1965) may rise still further during the acldi- tional deposition of cell-wall material that occurs just pi-ior to cell breakdown in this tissue (Schulz and Jensen, 19'71). Accompanying respiratory costs for import and synthesis


Figure 3. Light micrographs of cotton ovules sampled either 1 d before, the day of, or 1 d after flower opening/anthesisshowing areas of Sue synthase immunogold localization at low or expanded levels of magnification (37X to 368X). A andD, A cotton ovule 1 d before flower opening/anthesis showing extensive Sue synthase in nucellar and vascular tissues. Notethat little Sue synthase label is evident in epidermal tissues in either low (A) or expanded magnification (D) of the boxed areain A. B and E, A cotton ovule on the day of flower opening/anthesis showing dense immunolabeling of Sue synthase in thenucellus, facing epidermis of the inner integument, vascular tissues, and elongating hairs in either low (B) or expandedmagnification (E) of the boxed area in B. C and f, A cotton ovule 1 d after flower opening/anthesis showing the associationof Sue synthase with the nucellus, vascular tissues, and rapidly elongating epidermal hairs at low magnification (C). Thespecificity of gold label in association with the growing hairs is shown in the high-magnification light micrograph (F) of theboxed region in C. Bars in A through C = 250 ;u,m; Bars in D through F = 25 jum.

1290 Nolle et al. Plant Physiol. Vol. 109, 1995

B

*

Figure 4. (Legend appears on facing page.)


Anthesis

Intact Emasculated

« £«, "5 c! i ;* £ ±ra * w>« "5 >.S i- 3r- O T-

i I•° ™>• oW *ta.Q %i- O

SucroseSynthase

Figure 5. RNA gel blot analysis of Sue synthase transcripts fromovules of intact and emasculated cotton flowers. RNA gel blots withequal amounts (10 /ng) of total RNA isolated from ovules 1 d before,at, and 1 d after anthesis. Blots were probed with 32P-labeled Suesynthase cDNA clones (Sh 1 and Susl from maize) and exposed onx-ray film for 48 h.

could also be elevated in the developing nucellus and areconsistent with the presence of numerous mitochondria,elaborate plastids, and abundant ER (Jensen, 1965).

Callose formation and apparent assimilate transfer bythe nucellus and inner integument extend the probablefunction of Sue synthase in these tissues beyond those ofstorage-related processes alone. The megasporocytes ofmany angiosperm ovules are completely encased by dep-ositions of callose (Rodkiewicz, 1970; Schulz and Jensen,1971). This polysaccharide is also formed extensivelywithin both the nucellus and inner integument (Kapil andTiwari, 1978; Ryser et al., 1988). UDP-Glc is readily utilizedfor callose formation (Morrow and Lucas, 1987), and a closeassociation has been shown between the presence of Suesynthase and the potential for callose production in phloem(Geigenberger et al., 1993; Nolte and Koch, 1993).

Assimilate transfer and secretory surfaces of the inner-most integument (Ryser et al., 1988) and nucellus (Jensen,1965) would also be expected to show a Sue synthase linkto probable elevations in respiratory activity. The nucellarface of the integument in cotton (Ryser et al., 1988) and anumber of dicotyledonous species (Kapil and Tiwari, 1978)has features resembling the secretory/transfer cells of atapetum, including elaborate cell walls, extensive ER, mul-

tivesicular bodies, and a large number of well-developedmitochondria. Transfer functions of this tissue are reportedto persist later in seed development (Offler and Patrick,1993); however, a digestive function has also been pro-posed (Ryser et al., 1988). Nucellar cells can show some ofthe same structural features, but like the Sue synthase inthis tissue, these changes develop over a broader, lesswell-defined region (usually elongating cells closest to thenew embryo) (Jensen, 1965; Kapil and Tiwari, 1978).

A second goal of the present work was to address thebasis for differential rates of trichome initiation on oppositeends of the seed. These emerge first from the chalazalregion and later from the micropilar end (Figs. 2 and 3).Although reported previously (Basra and Malik, 1984;Ryser, 1985), the underlying factors remain unclear. Localdifferences in carbohydrate supply may be responsible.Structural information shows that phloem terminates nearthe most rapidly differentiating trichomes, and in otherspecies cells of the nucellus and integuments are signifi-cantly larger in areas surrounding vascular bundles(Schulz and Jensen, 1971; Sumner and Caesella, 1987). Inaddition, the Sue synthase localization in this region (Fig.2) is typical not only of sites where rapid assimilate importand transfer occur (Hawker and Hatch, 1965; Tomlinson etal., 1991), but also is consistent with up-regulation of Suesynthase genes by elevated sugar supplies (Susl in maize[Koch et al., 1992]; counterpart genes in other species [Sala-noubat and Belliard, 1989; Karrer and Rodriguez, 1992;Heim et al., 1993]).

The third and most striking portion of this work (Figs.2-4) shows that Sue synthase was closely associated withspecific, individual cells at the earliest visible stages in theirdifferentiation as trichomes. This became still more pro-nounced during subsequent elongation of these hairs. Theappearance and cell-specific immunolocalization of Suesynthase was found to clearly correlate with the first signsof trichome swelling as spherical epidermal outgrowths onthe day of flower opening/anthesis. This also coincidedwith the formation of a large central vacuole by the merg-ing of numerous smaller ones. In some instances (Fig. 4C),Sue synthase was immunolocalized to specific cells prior toany change in external features.

The most pronounced associations, however, were ob-served in rapidly expanding trichome cells (Fig. 4), whereSue synthase has been hypothesized to provide precursorsfor the metabolic demands of extensive cell-wall formation(Basra and Malik, 1984; Hendrix, 1990; Delmer and Amor,1995). Although the level of reducing sugars in developing

Figure 4. (On facing page.) Light micrographs of cotton ovules sampled either 1 d before, the day of, or 1 d after floweropening/anthesis showing areas of Sue synthase immunogold localization in epidermal cells at high magnification (920X).A, B, and C, Epidermal cells 1 d prior to flower opening/anthesis showing the degree of differentiation and Sue synthaseimmunoreactivity prior to initiation of rapid expansion in hair cells. Little or no Sue synthase protein was detected in cellswith well-defined nuclei and small vacuoles (A and B), whereas immunolabel was apparent in association with occasionalcells having enlarged central vacuoles (C). D, E, and F, Rapidly developing epidermal hairs on the day of floweropening/anthesis. Note the cytoplasmic and possible cell-surface localization of the immunogold label in these elongatingcells. G, H, and I, Expanding epidermal hairs 1 d after flower opening showing intense immunoreaction in the growingtrichome. Note the specificity of the gold label in association with developing hair cells relative to the adjacent nonex-panding cells (I). Bar = 50 /j,m.

1292 Nolte et al. Plant Physiol. Vol. 109, 1995

cotton fibers is relatively high during their elongation (Hendrix, 1990), the predominant sugar nucleotide is UDP- Glc during both primary and secondary wall formation (Carpita and Delmer, 1981). Other evidence also favors UDP-Glc as a precursor for synthesis of cellulose (consti- tuting about 30% of primary cell walls in cotton) (Meinert and Delmer, 1977; Carpita and Delmer, 1981; Delmer, 1987; Delmer and Amor, 1995) and for production of other UDP sugars used in formation of hemicellulose and pectin (Ami- no et al., 1985). Despite substantial activity of UDP-Glc pyrophosphorylase (an alternate source of UDP-Glc from Glc-1-P and UTP) (Wafler and Meier, 1994), Suc synthase is active in young ovules during fiber expansion (Hendrix, 1990) and provides an additional, more direct path of C transfer from symplastically imported Suc to UDP-Glc (Ryser, 1992; Delmer and Amor, 1995).

Such a role for elevated Ievels of Suc synthase would be consistent with the results described here. In some instances immunolocalization of this enzyme even appears to be enhanced in subcellular regions where most rapid expansion is taking place (Fig. 4, D and G). Labeling at the cell periphery, especially where elongation is most rapid, is compatible with the recent suggestion that some of the Suc synthase may be tightly associated with cellulose synthase in the plasma membrane (Delmer et al., 1995). Overall cytoplasmic labeling also appears to be heavier in these expanding portions of cells (particularly in Fig. 4D); however, this aspect of subcellular data must be viewed with caution because the thickness and extent of immunolabel can vary from end to end of an individual cell if a section is oblique.

At the whole-cell level, our data extend that obtained from isolated protoplasts during wall formation (Maas et al., 1990) and further implicate the importance of Suc synthase in supplying UDP-Glc during cell-wall biosynthesis. Additional requirements for products of the Suc synthase reaction in trichomes could also include provision of substrates for other growth-related processes such as increased respiratory demand (Farrar and Williams, 1990) and osmotic constituents for the turgor pressure supporting cell expansion (Cosgrove, 1986).

Pollination is a central regulatory process in flower development often affecting pigmentation and senescence of the perianth, ovary maturation, ovule differentiation, and gametophyte development (Woodson et al., 1992; Zhang and ONeill, 1993). Initiation of cotton fibers does not appear to be one of these, however. The present study shows that pollination is not required for either initiation or the earliest growth of trichomes, nor is it necessary for up-regulation of Suc synthase mRNA levels in these cells. In the work shown here, these processes proceeded similarly regardless of whether or not ovules had been pollinated (Fig. 5). Later stages in fiber development appear to be more dependent on pollination status, however, and do not take place in culture unless ovules are either fertilized or supplemented with additional auxin (Beasley and Ting, 1974).

The shift in cellular localization of Suc synthase during initiation of trichome development indicated a close association between Suc synthase and Suc import at a cellular

level in developing seeds. The results reported here are also consistent with the hypothesis that Suc synthase has a pivotal function in cell-wall biosynthesis in these fibers and provides substrates for other growth-related processes such as increased respiratory demand, osmotic adjustment, and/or callose formation (as in the nucellus and inner integument).

ACKNOWLEDCMENTS

The authors thank D.R. McCarty and L.C. Hannah for their assistance in aiitibody production, and J. Vinci for her help in preparation of !severa1 sets of material.

Received May 4, 1995; accepted August 10, 1995. Copyright Clearance Center: 0032-0889 /95 / 109 / 1285 / 09.

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