A floral transmitting tissue-specific glycoprotein attracts pollen tubes and stimulates their growth

Cell, Vol. 82, 383-393, August 11, 1995, Copyright© 1995 by Cell Press

A Floral Transmitting Tissue-Specific Glycoprotein Attracts Pollen Tubes and Stimulates Their Growth Alice Y. Cheung, Hong Wang, and Hen-ming Wu Department of Biology Yale University New Haven, Connecticut 08520-8104

Summary Pollen tubes elongate directionally in the extracellular matrix of pistil tissues to transport the male gametes from the apically located stigma to the basally located ovary for fertilization. The molecular mechanisms underlying directional pollen tube growth in the pistil are poorly understood. We have purified a glycoprotein, TTS, from tobacco stylar transmitting tissue, which supports pollen tube growth between the stigma and the ovary. TTS proteins belong to the arabinogalactan protein family, and they polymerize readily in vitro in a head-to-tail fashion into oligomeric forms. TTS proteins stimulate pollen tube growth in vitro and attract pollen tubes grown in a semi-in vivo culture system. In vivo, the pollen tube growth rate is reduced in transgenic plants that have significantly reduced levels of TTS proteins as a result of either antisense suppression or sense cosuppression. These results identify TTS protein as a pistil component that positively contributes to pollen tube growth. Introduction The male and female gametes in flowering plants are spatially separated. The male gametes reside in the pollen that is deposited on the pollen-receptive stigmatic surface of the pistil (the female reproductive organ). Each pollen grain germinates and extrudes a pollen tube that penetrates the stigma and elongates through the style to reach the ovary. Once inside the ovary, each pollen tube enters an ovule and penetrates the embryo sac, bursts, and releases the male gametes for fertilization. Successful fertilization depends on specific pollen-pistil interactions, and only compatible pollen grains are able to complete their passage (Knox, 1984). Pollen tube elongation is a tip growth process in which the tube cell cytoplasm, nucleus, and the two sperm cells are confined to the tip of the tube (Heslop-Harrison, 1987; Steer and Steer, 1989). Pollen tube growth takes place in the extracellular matrix (ECM) of the stigmatic and stylar transmitting tissues and along the ovule surface. As the pollen tube extends, callose (1~-1,3-glucan) plugs are periodically formed behind the cytoplasmic zone, blocking it from the noncytoplasmic region of the tube. Pollen tube growth has been described as a specialized form of plant cell movement in which the pollen cytoplasm moves forward, leaving behind a trail of cell wall materials connecting the tube tip to the empty pollen grain that remains anchored on the stigmatic surface (Lord and Sanders,

1992). The extension of the pollen tube tip is often compared with other tip growth processes, such as hyphal growth in fungi, root hair elongation in plants, and neuronal axon outgrowth in animals (Heath, 1990). The pistil ECM is believed to provide chemical and physical supports as well as directional cues for pollen tube extension toward the ovules (Mascarenhas, 1975, 1993; Heslop-Harrison, 1987; Lord and Sanders, 1992). In this sense, pollen tube growth is most similar to axon outgrowth in which the growth cone interacts with ECM molecules and responds to chemotropic agents emanating from their target sites to attain directed growth (Tessier-Lavigne and Placzek, 1991 ; Hynes and Landers, 1992). Pollen has a high capacity to support its own activity during germination and tube growth (Steer and Steer, 1989; Vasil, 1987). However, the pistil must also contribute to this process, since in vitro grown pollen tubes extend in random directions, their growth rates are usually lower, and they reach considerably shorter distances than in vivo pollen tubes. Yet, little is known about the pistil components that may contribute to compatible pollen tube growth (Cheung, 1995). The transmitting tissue in the style is a specialized tissue through which pollen tubes elongate from the stigma to the ovary. Its ECM is enriched with secretory materials, such as free sugars, polysaccharides, amino acids, giycoproteins, and glycolipids. Arabinogalactan proteins (AGPs), which are ubiquitous to plants and are believed to have numerous functions in plant growth, development, and cell-cell interactive processes (Showalter, 1993; Kreuger and van Hoist, 1993; Serpe and Nothnagel, 1994), constitute a major class of proteins in the ECM of the transmitting tissue and in the stigma exudates (Clarke et al., 1979b; Gleeson and Clarke, 1980). Their abundance in the pistil as well as their stickiness and high sugar contents has prompted speculations that these proteins may be involved in pollen recognition and adhesion on the stigma and that they may serve as nutrients and adhesive substrates for the growing pollen tubes in the transmitting tissue (Labarca and Loewus, 1973; Clarke et al., 1979a, 1979b; Lord and Sanders, 1992). Several transmitting tissue-specific cDNAs for prolinerich proteins have recently been isolated and characterized (e.g., Chen et al., 1992; Goldman et al., 1992; Cheung et al., 1993). Two closely related cDNAs, TTS-1 and TTS-2, from tobacco encode proline-rich glycoproteins located specifically to the ECM of the transmitting tissue (Wang et al., 1993). We report here the purification and characterization of TTS proteins and show that they belong to the AGP family, attract and stimulate pollen tube growth in vitro, and are needed for optimal in vivo pollen tube growth.

Results Purification of TTS Proteins TTS proteins have been purified from citric acid extracts eluted from hand-bisected styles (Figu re 1A). TTS proteins

Cell 384

Table 1. SugarAnalysisof TTS Proteins

1 2 345678 -203 -108 -70 -43 -27 B t 234M5 -2O3 -105 -70 It

Sugars

Micrograms of Sugar/100~g of Polypeptide" (Percent of Total Sugars)

Galactoseb Glucosec Galaturonicacid Arabinose~

35.3 (70) 8.4 (17) 3.25 (6.5) 3.1 (6)

"Mass of polypeptide.Totalproteinmass is the sum of sugarcontent and polypeptide. b32% of the galactoseresidueswereterminal,6% were3-1inked,28% were 6-1inked,15% were 2,3-1inked,and 13% were 3,6-1inked. c6-1inked. d68% of the arabinoseresidueswere terminal, 15% were 2-1inked, 15o/owere 5-1inked, lO/owas 2,3-1inked.

-43 -27

Figure 1. Purification and Characterization of TTS Proteins

(A) Purificationof TTS proteins.Lanes1-4, silver-stainedSDS-PAGE (12.5%)of 2 ~g of total pistil proteins(lane1), acidbuffer-elutedtransmitting tissue ECM proteins(lane 2), G100 fraction (lane3), or CMSepharose fraction (lane 4). Lanes 5-8, immunoblotsof the same samples shown in lanes 1-4, respectively,exceptthat 20 #g of total pistil proteinswas loadedin lane 5. (B) Characterizationof TTS proteins. Lanes 1 and 2, silver-stained SDS-PAGE(12.5%)of chemicallydeglycosylatedTTS proteins(1 #g; lane 1) and nativeTTS proteins(2 ~.g;lane2). Lanes3 and 4, immunoblotof nativeTTS proteins(2 ~.g;lane3) and chemicallydeglycosylated TTS proteins(1 p.g;lane 4). Lane 5, proteinblot of nativeTTS proteins(2 ~.g)stainedwithYarivreagent.M, prestainedproteinmolecular weight markers. The proteinblots werereactedwith TTS antibodiesand alkalinephosphatase-labeledsecondaryantibodies.The numbersto the right of the figuresare molecularweights in kilodaltons.

have a broad apparent molecular weight spectrum of between 50-100 kDa on SDS-polyacrylamide gels. Chemical deglycosylation reduced the heterogeneous proteins to molecular weight species of around 30 kDa (Figure 1B, lanes 1 and 2), close to the deduced molecular weights for TTS polypeptide backbones (Cheung et al., 1993). No other protein bands were detected by silver staining of the purified proteins or their deglycosylated forms after SDSpolyacrylamide gel electrophoresis (SDS-PAGE), indicating that the proteins were close to homogeneity. Antibodies to TTS proteins recognized the 50-100 kDa proteins as well as the 30 kDa deglycosylated species (Figure 1B, lanes 3 and 4). The molecular weight heterogeneity of the purified proteins is characteristic of many plant hydroxyproline-rich glycoproteins, especially AG Ps. The relatively broad deglycosylated 30 kDa protein band was a result of the slight length and composition heterogeneity between their protein backbones, low levels of residual sugar moieties, and the relatively large amounts of proteins analyzed. Amino acid composition and partial peptide sequence analyses confirmed that the purified proteins are TTS proteins (data not shown). The $20 values of TTS proteins center around 3.9, reflecting an average molecular mass of around 50 kDa. The considerably higher apparent molecular weights (between 50-100 kDa) determined by SDS-PAGE (Figure 1) probably resulted from anomalous electrophoretic properties due to sugar modifications.

Amino acid analysis showed that 57% of the proline residues in TTS proteins are hydroxylated. Sugar residues constitute about 35% of the total TTS protein molecular mass (Table 1). TTS proteins react strongly with a ]3-glucosyl Yariv dye (Figure 1B, lane 5), a diagnostic reagent for AGPs (Yariv et al., 1967), indicating that they belong to the AGP family. Besides being proline rich, the deduced amino acid sequences of TTS proteins (Cheung et al., 1993) share little homology with those of the other two AGPs reported to date (Chen et al., 1994; Duet al., 1994). However, TTS-related mRNAs and proteins are present in several other solanaceous plants, in Arabidopsis, maize, and lily (data not shown). A cDNA homologous to TTS cDNAs has been reported in Nicotiana alata (Chen et al., 1993). Electron microscopic images of rotary-shadowed TTS proteins show that monomeric TTS molecules are round to slightly elliptical (Figure 2) and are about 30 nm in diameter. The structure closely resembles the "wattle-blossom" model proposed for AGPs (Fincher et al., 1983) and that of a carrot AGP (Baldwin et al., 1993). TTS molecules polymerize into various highly ordered oligomeric forms with no free ends. The high molecular weight aggregates are made up of congregations of smaller oligomers of between 10-15 TTS molecules (Figure 2G). This is consistent with the tendency of TTS proteins to aggregate during purification and storage and with the known adhesive property of AG Ps. TTS Proteins Stimulate Pollen Tube Gre.wth In Vitro Tobacco pollen grains germinated adequately, and their tubes were morphologically normal for at least the first 8-10 hr in a sugar-free pollen germination medium (Figure 3A). Pollen tubes elongated with an average rate of 50 i~m/hr during the first 6 hr of growth (Figure 3C). Addition of purified TTS proteins enhanced the pollen tube growth rate (Figure 3B), and this stimulation increased linearly between 0.2-2 p.g/ml TTS proteins (Figure 3C). At 2 p.g/ ml TTS proteins, the average pollen tube growth rate was 150 ~m/hr, constituting an approximate 3-fold stimulation from that observed in unsupplemented cultures. Addition of similar concentrations of bovine serum albumin did not have stimulatory effects on pollen tube growth (data not

Pollen Tube Growth Promoter and Attractant 385

D

Figure2. Electron Micrographs

of Rotary-

Shadowed TTS Proteins (A) Monomeric and oligomeric TTS proteins. (B) Monomers.(C) Dimer.(D)Trimers.(E) Dimer and tetramer. (F) Pentamers and hexamers. (G) Multimer. Scale bars represent 100 nm.

C

:::.

shown). The pollen tube growth-stimulating activity increased only slightly between 2-5 i~g/ml TTS proteins and reached a plateau at 10 ~g/ml TTS proteins (Figure 3C). It dropped precipitously when the TTS protein concentration reached 100 p.g/ml when TFS proteins began to aggregate. Since sugar residues constitute about 35% of the total molecular mass of TTS proteins (Table 1), the sugar concentration in 2 p.g/ml TTS proteins is 0.7 ~g/ml. When 2 ~g/ml TTS proteins and 2 mg/ml of sucrose, glucose, gatactose, or arabinose were added to pollen tube growth cultures, the stimulatory effect of TTS proteins on pollen tube elongation persisted when compared with cultures supplemented with these sugars only (Figures 3D and 3E), although the promotion was reduced to about 1.5fold. When chemically deglycosylated TTS proteins were added to these in vitro cultures, no pollen tube growth stimulation was observed (data not shown). However, it was not known whether other protein properties had been affected by the deglycosylation reaction. TTS Proteins Attract Pollen Tubes in S e m i - I n Vivo Pollen Tube Cultures A semi-in vivo pollen tube growth system (Kandasmy and Kristen, 1987) has been modified to exam ine whether TTS proteins attract pollen tubes. A pollinated pistil is cut close to the bottom of the style and cultured with the cut end immersed in a pollen tube growth medium. Pollen tubes whose tips have not reached the cut site will emerge from the cut end and continue to elongate in the medium. This system is most suitable for assays of attractants or repellants for pollen tubes, since they emerge from the style in one direction. This is impossible to accomplish in systems involving in vitro pollen germination that always result in pollen tubes emerging in all directions. Moreover, the pollen tubes emerging from these styles are likely to be more sensitive to exogenous growth supplements than freshly

germinated pollen tubes that may utilize reserves in the pollen grains. Pollen tubes that emerge from these styles continue to grow on this medium, and they are morphologically normal and have regular deposition of callose plugs as they elongate (Figures 4G and 4H). When observed at about 20 hr after either control or TTS proteins had been implanted at a distance from the pollen tube front, pollen tubes on control slides have extended away from the cut style into a fan-shaped mesh (Figures 4B, 4G, and 41). TTS proteins implanted to one side of the pollen tube front attracted these tubes (Figures 4C-4E and 4H). When control and TTS protein plugs were implanted on either side of the styles, pollen tubes were preferentially attracted by TTS proteins (Figure 4J). Free sugars, e.g., sucrose (Figure 4K), were not as effective as TTS proteins in attracting pollen tubes. When implanted in a medium supplemented with 0.1% sucrose, TTS proteins continued to attract pollen tubes (Figure 4F), although the sensitivity of these tubes toward -I-I'S proteins was less pronounced than when they were cultured in the sugar-free medium. However, chemically deglycosylated TTS proteins were not effective in attracting pollen tubes (Figures 4L and 4M). Transgenic Plants with Highly Reduced TTS Protein Levels Have Reduced Female Fertility Antisense suppression is well documented in transgenic plants (e.g., Smith et al., 1988). Introduction of a transgene in the sense orientation occasionally leads to cosuppression of the endogenous gene(s) and the transgene (Jorgensen, 1990). We examined 85 tobacco plants transformed with a TTS gene promoter:antisense TTS transgene (antiTT3) and 40 plants transformed with a CaMV 35S promoter:sense TTS transgene (35STTS). These plants were vegetatively and florally indistinguishable from control plants. The TTS mRNA and protein levels in these plants

Cell 386

A

B

5~ i

0

g

S

C

micrometers/ hour 150

T

125 100 75 5O

'1

'2

'3

'5";0

micrograms "11'Sprotein/ml

D ° P q

!

have been analyzed (Figures 5A-5C): 92 of these transgenic plants maintain relatively normal levels of TTS mRNAs and proteins and are, thus, referred to as normal TTS plants; 13 transgenic plants (12 anti-TTS and 1 35STTS) have between 2 5 % - 5 0 % of the control levels of TTS mRNAs and proteins (e.g., anti-TTS-lO) and are designated moderate TTS plants; 20 transgenic plants (18 antiTTS and 2 35STTS) have significantly reduced TTS mRNA and protein levels (<25% of the levels in control plants), e.g., 35STTS-J, anti-TTS-2, anti-TTS-3, anti-TTS-12, and anti-TTS-26, and are designated low TTS plants. The level for another transmitting tissue-specific proline-rich protein mRNA (Goldman et al., 1992) is not affected in any of these plants (data not shown), indicating that the suppression is not a general phenomenon. After natural self-pollination, all of the normal and moderate -I-FS plants have fruit and seed yields comparable to those produced by control plants (e.g., anti-TTS- 10, Figu re 5D; 35STTS-N, anti-TTS-lO, and anti-TTS-15, Table 2). Three of the low TTS plants (35STTS-J, anti-TTS-2, and anti-TTS-3) produce very small fruit capsules and have low seed yields (<10% of control plants) (Figure 5D; Table 2). Very often, flowers on these three plants abscise at about 3 - 4 days after anthesis, indicating that fertilization has not occurred. These three plants are referred to as low TTS RF plants (RF, for reduced fertile). The remaining 17 low TTS plants have mild reproductive phenotypes, producing seed yields of between 6 0 % - 9 0 % of those from control plants, e.g., anti-TTS-12 (Figure 5D; Table 2), indicating some impairment in the reproductive process (see below). The TTS mRNA and protein levels, and the reproductive phenotypes described above, are retained in the second generation transgenic plants that have been examined, indicating that they are not artifacts arising from transformation conditions. When pollen from these transgenic plants was used to pollinate wild-type stigmas, normal fruit size and seed yields were obtained (W38 x 35STTS-J and SR1 x antiTTS-3, Figure 5D; "uninterrupted" column, Table 3). This indicates that the reduced fertile phenotype associated with the low TTS RF plants is a female property. T h e P o l l e n T u b e G r o w t h R a t e Is R e d u c e d in the L o w T T S P l a n t s

E

G

o

Figure 3. TTS ProteinsStimulate Pollen Tube Growth In Vitro (A and B) Pollentubesweregrownin a sugar-depletedgrowthmedium supplementedwith 0 ~.g/ml(A) or 2 p_g/ml(B) of "I-I'S proteinsfor 6 hr. (C) Averagepollen tube elongation(micrometersper hour)was determined from four independent pollen tube growth experimentswith

The pollen tube growth rate in the low TTS plants was determined by a direct and an indirect method and found to be reduced relative to that in control plants. Handpollinated pistils were excised at a time when pollen tubes were reaching the bottom 0.5 cm of the wild-type styles (segment 7, Figure 6A) and treated for direct microscopic

different preparationsof TTS proteins.Pollentube cultures in a sugarfree medium were supplementedwith 0, 0.5, 1, 1.5, 2, 3, 5, and 10 t~g/mtTTS proteins and incubatedfor 6 hr. (D and E) Pollen tubes were grown in mediumsupplementedwith 2 #.g/mlgalactose(D), or 2 mg/ml galactoseand 2 mg/mtTTS proteins (E) for 3 hr. The averagelengths of these pollen tubes were 510 ~m and 690 #m in (D) and (E), respectively.Similarlevelsof stimulationby TTS proteinswere obtainedwhenthe sameconcentrationsof glucose, sucrose, or arabinosewere used (data not shown). Scale bars represent 250 p.m.

Pollen Tube Growth Promoter and Attractant 387

Figure 4. ITS ProteinsAttract Pollen Tubes in Semi-In Vivo Pollen Tube Growth Cultures (A) Pollen tubes emerging from a cut style at 10 hr after culturing on a sugar-free medium when agarose plugs of control or TTS proteins were applied. Sam pies in (B)-(M) were cultured for about 20 hr after these applications (the approximate locations of agarose plugs are indicated by white arrows when they are within the view of the micrographs). (B) Pollen tubes growing with sugar-free medium implanted at 1.5 mm to the right of the style. (C) Pollen tubes growing with TTS proteins (0.2 I~g/l~l) implanted at 1.5 mm to the right of the style. (D and E) Pollen tubes growing with TTS proteins (0.5 pg/#l) implanted at 3 and 4 mm, respectively, to the right of the style. (F) Pollen tubes growing in medium containing 0.1% sucrose and with TTS proteins (0.2 ~g/ I~l) implanted at 2 mm to the right of the style. (G) Aniline blue-stained control pollen tubes. (H) Aniline blue-stained pollen tubes growing with TTS proteins (0.2 I~g/F,I)implanted at 2 mm to the right of the style. (I) Pollen tubes growing with sugar-free medium implanted at 3 mm from both sides of the style (not included in the view). (J) Pollen tubes growing with TTS proteins (0.2 ~g/p.I) implanted at 3 mm to the right of the style, and sugar-free medium implanted at 3 mm to the left of the style. (K) Pollen tubes growing with TTS proteins (0.2 14g/l~l) implanted at 3 mm te the right of the style, and medium with 0.1% sucrose implanted at 3 mm to the left of the style. (L) Pollen tubes growing with chemically deglycosylated TTS proteins (0.2 #g/~_l) implanted at 3 mm to the right of the style, and sugar-free medium at 3 mm to the left of the style. (M) Pollen tubes g rowing with TTS proteins (0.2 ~g/p_l) implanted at 3 mm to the right of the style, and chemically deglycosylated TTS proteins (0.2 pg/~l) at 3 mm to the left of the style. Scale bar in (M) equals 1 mm and applies to all micrographs except (G) and (H). Scale bars in (G) and (H) equal 1.6 mm. observation of pollen tubes (Figure 6A, top left). In the low TTS RF plants, e.g., anti-TTS-3, no pollen tubes were observed in the bottom segment (7) of these styles (data not shown). Instead, a very low number of pollen tubes were observed in segment 6, between 0.5-1 cm a b o v e the s t y l e - o v a r y junction (Figure 6A, bottom right). In the styles of the low TTS plants, e.g., anti-TTS-12, only a few pollen tubes had reached s e g m e n t 7 (Figure 6A, top right). Increasing numbers of pollen tubes were observed in stylar segments incrementally closer to the stigma of these plants (e.g., c o m p a r e segments 2, 5, and 6 of the antiTTS-3 style, Figure 6A, bottom), indicating that the n u m b e r of pollen tubes penetrating these transgenic styles did not differ appreciably from that in the control styles. These results indicate that the pollen tube growth rate was reduced to various extents in transgenic plants with very low TTS protein levels, and the most significant reduction was observed in the low TTS RF plants. When pollen from these transgenic plants, e.g., anti-TTS-3, cross-pollinated

control styles, c o m p a r a b l e n u m b e r of pollen tubes were observed in the bottom stylar segment (7) (Figure 6A, top middle) as in self-pollinated control styles (Figure 6A, top left), suggesting that a pollen-associated tube growth phenotype was not obvious in these transgenic plants. A similar conclusion that the pollen tube growth rate was reduced in transgenic plants with very low levels of TTS proteins could be made when the growth rate was assessed indirectly (Figure 6B; Table 3). In this assay, handpollinated styles were excised at 0.5 cm a b o v e the o v a r y style junction at a time when pollen tubes were beginning to enter the ovary in control plants. The ovaries were left on the plant for fruit and seed development. This treatment allowed pollen tubes that had extended beyond the cut site to continue their growth and to enter and fertilize the ovules, but it precluded the slower pollen tubes whose tips were a b o v e the cut site from reaching the ovary. Seed yields in control plants increased with longer intervals between the time of pollination and stylar excision (Figure

Cell 388

A

SR12 3 10 12 26 W38 J

B

SR12 3 10 12 26 W38 J

Table 2. Seed Yields from Control and Transgenic Plants" with Reduced TTS Protein Levels after Natural Self-Pollination

-105 -70 -43 C

SR12 3 10 12 26 W38 J

43 '27

~,~38

O SR1

Auti[ [S-2

35STTS.J

• AntiT] S 10

AntiTTS t~

W38 X 35sTrs-J

m An~l

2

-

Plant

Seed Yield (mg)c

Percent of Control

W38

252

100

J (low, RF)b S (low)

20 5-158 ~

N (normal)

240

SR1 2 (low, RF) 3 (low, RF) 12 (low)e 26 (low) 10 (moderate) 15 (normal)

149 9.8 3.6 98 135 150 145

7.9 2-62 95 100 6.5 2.4 66 90 100 97

"J, S, and N are 35STTS plants and are W38 derivatives. All the other plants are anti-TTS plants and are SR1 derivatives. b (Low, RF), (low), (moderate), and (normal) designate low TTS RF plants, low TTS plants, moderate TTS plants, and normal TTS plants, respectively. c Averaged from ten fruit capsules. d The 35STTS-S plant showed a range of fertility. e Data from representative low, moderate, and normal TTS plants are shown.

SRI XAr~a] TS 3

Discussion Figure 5. Transgenic Tobacco Plants with Highly Reduced TTS mRNA and Protein Levels Show Reduced Female Fertility (A) mRNA blot showing the levels of TTS mRNAs from representative transgenic plants. W38 is the control for the transgenic plant 35STTS-J (J), and SR1 is the control for the other transgenic plants anti-TTS-2 (2), anti-TTS-3 (3), anti-TTS-lO (10), anti-TTS-12 (12), and anti-TTS-26(26). (B) Protein blot showing the levels of TTS proteins from the same transgenic plants as in (A). Plants J, 2, 3, 12, and 26 belong to the low TTS plant group, and plant 10 is an example of a moderate TTS plant. (C) Protein blot for the same plants shown in (B), but the samples have been chemically deglycosylated. (D) Fruit capsules and seed yields resulting from natural self-pollination of control and selective transgenic plants. The fruit capsules from crosses W38 x 35STTS-J (top right) and SR1 x anti-TTS-3 (bottom right) were from wild-type pistils that had been pollinated by the two transgenic plants.

6B; Table 3). In the low TTS RF plants, most of the ovaries abscised after similar treatments, indicating that fertilization had not occurred. Fruits that had developed were highly atrophied, and seed yields were very low (Figure 6B; Table 3). When pollen tube growth was left uninterrupted, differences in seed yields between the control and these transgenic plants were minimized, although these transgenic pistils continued to produce lower amounts of seeds. Results were similar when these experiments were repeated in the second generation transgenic progeny of these plants. Seven of the low TTS plants were similarly analyzed. Their seed yields were all lower than in control plants when pollen tube growth was interrupted (between 2 3 % - 7 2 % of the yields in control plants) (Table 3). These results are consistent with those from the direct visualization of pollen tubes (Figure 6A) and indicate that the pollen tube growth rate in these low TTS plants is reduced, but not as significantly as in the low TTS RF plants.

Pollen tubes often extend for distances hundreds to thousands of times that of the pollen grains to reach the ovary. Copious amounts of cell wall materials, much of which are carbohydrate-containing molecules, are synthesized during this process. Therefore, sugar molecules are a much needed resource for pollen tube growth and are known to e n h a n c e this process in vitro. Pollen tube growth occurs in the ECM of pistil tissues, and surface adhesive molecules have been proposed to be important for this process (Lord and Sanders, 1992). We show in this paper that a transmitting tissue ECM glycoprotein from tobacco, TTS, stimulates pollen tube growth when evenly distributed in in vitro cultures and attracts pollen tubes when provided from a distance in s e m i - i n vivo cultures. When TTS proteins are reduced to very low levels in transgenic plants, the in vivo pollen tube growth rate is reduced. Taken together, these results indicate that TTS proteins function to e n h a n c e pollen tube growth.

TTS Proteins Belong to the AGP Family AGPs are ubiquitous plant hydroxyproline (or proline)-rich proteins with characteristic sugar linkage properties. The properties of TTS proteins (Table 1 ; Figure 1) allow them to be categorized into the AGP family. However, the level of sugar modifications in TTS proteins is low relative to other known AGPs in which sugar moieties may constitute more than 9 0 % of their molecular mass. Furthermore, TTS proteins are basic, a characteristic distinct from several other known AGPs. As more galactose- and arabinose-rich proteins with A G P properties are being characterized (Kieliszewski et al., 1992; Baldwin et al., 1993; Lind et al., 1994) and their amino acid sequences deduced (Cheung et al., 1993; D u e t al, 1994; Chen et al., 1994), it is apparent that there may be significant structural and chemical vari-

Pollen Tube Growth Promoter and Attractant 389

Table 3. Seed Yields from Control and Transgenic Plants~with Reduced TTS Protein Levels after Interrupted Pollination Seed Yield, mgc (Percent of Control) Female Parent

Male parent

26 hrd

28 hru

W38 J (low, RF)b W38 SR1 2 (low, RF) 3 (low, RF) 12 (low)' 26 (low) SR1

W38 W38 J SR1 SR1 SR1 SR1 SR1 3 (low, RF)

40 (100) 11 (27) 37 (93)

49 (100) 20 (40.8) 50 (102)

31 hru

22 (100) 2.1 (9.3) <10 seeds 5.1 (23) 10.9 (49) 23 (104)

33 hr~

Uninterruptede

45 (100) 6.3 (14) 4.7 (10.4) 12.6 (28) 25.6 (57) 43 (95)

250 (100) 109 (42) 238 (97) 160 (100) 57 (36) 30 (19) 134 (84) 162 (101) 152 (95)

aj is a 35STTSplant and is a W38 derivative. All the other plants are anti-TTS plants and are SR1 derivatives. b (Low, RF) and (low) designate low TTS RF plants and low TTS plants, respectively. ° Averaged from ten fruit capsules. d Indicates the length of time pollen tube growth proceeded before the styles were excised at 0.5 cm above the style-ovary junction. e Styles were excised at 72 hr after pollination, just shortly before they would normally abscise. The seed yields from these hand-pollinated flowers were consistently higher than the naturally self-pollinated flowers. This was probably due to the very large load of pollen grains synchronously applied by hand-pollination. Despite reduced growth rates, higher numbers of pollen tubes must have entered the ovary before the style abscised than attainable by natural pollination. f Data from two representative low TTS plants are shown. The percent seed yields from the other five plants examined relative to the control after interrupted pollination ranged between 30%-72%.

ability among this family of proteins. Although the functional significance of TTS proteins does not lie in whether they belong to the AGP family, the fact that they do affirms previous proposals that AGPs are important to pollen tube growth (Clarke et al., 1979a, Fincher et al., 1983; Lord and Sanders, 1992). The oligomeric structures observed for TTS proteins in vitro (Figure 2) indicate that their monomers tend to polymerize linearly in a head-to-tail fashion. We speculate that prolonged linear polymerization of TTS protein molecules is not favorable in the absence of surface support in vitro, and so these linear polymers circularize to form the observed, energetically more favorable oligomers with no free ends. The N-terminal half of TTS proteins is hydroxyproline- and lysine-rich, and their C-terminal half is cysteine- and lysine-rich. Since hydroxyproline residues are the target sites for glycosylation (Showalter and Varner, 1989), the majority of sugar modifications in TTS proteins should be in the N-terminal half of these molecules. Glycosylation of TTS proteins acidifies them (Wu et al., 1995 [this issue of Cell]). Thus, the primary structure of TTS proteins may be bipolar with a very basic domain in the C-terminal half and a relatively less basic domain in the N-terminal half. Such a bipolarity might underlie charge interactions between the N- and C-termini of these molecules in the formation of TTS polymers. In vivo, whether TTS proteins polymerize in a similar head-to-tail manner along the transmitting tissue ECM into long chain molecules remains to be determined.

TTS Proteins Are Transmitting Tissue ECM Components Important for Pollen Tube Growth After pollen germination, tobacco pollen tubes extend in the stigmatic zone at a rate of 0.5 mm/hr. The growth rate increases abruptly to about 1.5 mm/hr once pollen tubes enter the transmitting tissue (Wang et al., submitted). A biphasic pollen tube growth characteristic has also been

observed in the pistil of other plants (Mulcahy and Mulcahy, 1982), and this has led to the suggestion that pollen tubes change from an autotrophic to a heterotrophic mode of growth within the style. The stimulation of pollen tube growth in vitro by TTS proteins (Figure 3), and the reduced pollen tube growth rates in transgenic plants with very low levels of these proteins (Figure 6), both strongly suggest that the functional role of TTS proteins is to enhance pollen tube growth within the transmitting tissue. TTS proteins do not accumulate in the stigmatic zone but are maintained at very high levels throughout the stylar transmitting tissue (Wang et al., 1993). Therefore, the higher pollen tube growth rate upon entering the transmitting tissue may at least partially be the result of stimulation by TTS proteins. In directional cell growth systems, promoters of growth often also attract cells along increasing concentrations of these substances. For example, nerve growth factor stimulates axonal outgrowth and can act as an attractant for regenerating axons in vitro (Gundersen and Barrett, 1979). Recently, two related proteins, netrin-1 and netrin-2, from embryonic chick brain have been shown to promote commissural axon outgrowth (Serafini et al., 1994). Recombinant forms of netrins secreted from transformed culture cell lines attract these axons from a distance (Kennedy et al., 1994). TTS proteins are both pollen tube growth promoters and pollen tube attractants (Figures 3 and 4). The growth of pollen tubes in the semi-in vivo cultures is along an increasing TTS protein concentration gradient that has resulted from diffusion from the source. Since TTS proteins are glycosylated, an increasing sugar concentration gradient coexists with the TTS protein gradient. Whether pollen tubes respond to the protein, the sugar gradient, or both remains to be resolved. Similarly, whether the pollen tube growth stimulatory activity of TTS proteins is based on their sugar contents or other properties associated with these proteins also remains to be determined. However, the decline of pollen tube growth stimulation by TTS proteins in the presence of a free sugar source (Fig-

Cell 390

WT9 × w ~

~9

(7)

(7)

ANTI3 +O X WTO~ (2)

ANTI-3 ~ X WT O~

ANTI-3 (~ X WT O~

(5)

26 hrs

B

x ,.'n-3 ¢a

28hrs

(6)

Uninterrupted

!B 0

35S TTS-J

31 hrs

33 hrs

Uninterrupted

SR1

Anti TTS-3

Figure 6. Pollen Tube Growth Rate Is Reducedin Transgenic Plants with Significantly Reduced Levels of TTS Proteins (A) Pollentube growthratein control and transgenicplantsas revealed by anilineblue staining.The female and maleparents in thesepollinations and the stylar segmentexaminedare indicated at the bottomof each photograph. (B) Fruitcapsulesand their seedcontentsfrom hand-pollinatedcontrol and transgenicpistils after pollen tube growth had been interrupted. The times 26 or 28 hr (for W38 and 35STTS-J) and 31 or 33 hr (for SR1 and anti-TTS-3) indicatethe time allowedfor pollen tube growth beforethe pollinatedstyleswere cut. Uninterruptedindicatesthat the styles were cut at 72 hr after pollination.All pollinationswere carried out by wild-typepollen grains. The female parents were indicatedon the left of each row.

ures 3D and 3E) and the inability of chemically deglycosylated TTS proteins to stimulate pollen tube growth or to attract pollen tubes (Figures 4K and 4L) suggest that the sugar moieties on TTS proteins are an important component of these activities, presumably by serving as nutrients. On the other hand, the persistence of a lower level of pollen tube growth-promoting activity (Figures 3D and 3E) and of the pollen tube-attracting activity from TTS proteins despite the presence of free sugars (Figures 4F and 4K) suggest that properties other than sugar provision also contribute to these two activities. The loss of these activities by chemical deglycosylation is not in conflict with this conclusion, but suggests that the sugar residues on TTS proteins are needed to mediate the nonnutrient providing aspect(s) of the TTS protein activities. The accompanying paper describes how TTS proteins interact with pollen tubes and provides some insights into the molecular bases underlying the pollen tube growth-stimulating and pollen tube-attracting activities of TTS proteins (Wu et al., 1995). Low molecular weight organic and inorganic chemicals, e.g., sugar, polyethylene glycol, and Cu 2÷, have been shown to have positive effects on pollen tube growth rates and on the quality of pollen tubes in in vitro cultures (Vasil, 1987; Read et al., 1993). Furthermore, Ca 2+ and glucose have also been shown to attract pollen tubes from a number of plant species in vitro (Mascarenhas and Machlis, 1962a; Reger et al., 1992a, 1992b). However, pollen tube growth-stimulating and -attracting activities have not been observed for any purified protein species, although crude and partially purified pistil tissue components from several plant species have been shown to enhance or to attract pollen tube growth (Rosen, 1961 ; Mascarenhas and Machlis, 1962b; Welk et al., 1965; Reger et al., 1992a). In this report, in vitro, semi-in vivo, and in vivo experiments have been performed to corroborate all the observations and establish a functional role for TTS proteins. All the results are consistent with a role of TTS proteins in promoting pollen tube growth.

Pollen Tube Growth Rate Is an Important Aspect of Plant Sexual Reproduction In wild-type tobacco plants, pollen tubes usually reach the ovary between 24-30 hr after pollen germination. The styles abscise around 72 hr after pollination, leaving the ovaries on the plant to develop into fruits. During natural pollination, some pollen grains are deposited on the stigma earlier than others, and some germinate more readily than others. The experiments involving the transgenic low TTS plants (Figures 5 and 6) showed that reproductive yields will drop if the pollen tube growth rate falls too low, such as in the low TTS RF plants. Presumably, most of the pollen tubes in the styles of these low TTS RF plants cannot reach the ovules before stylar abscission, resulting in reduced seed sets. On the other hand, these experiments also showed that plants have substadtial flexibility in accommodating variant pollen tube growth rates so that fertility is not significantly affected by slightly reduced growth rates, such as in the low TTS plants. Presumably, when pollen tube growth was not disturbed in

Pollen Tube Growth Promoter and Attractant 391

t h e s e plants, e n o u g h pollen t u b e s a r r i v e d at t h e o v a r y to fertilize a high p e r c e n t a g e o f the o v u l e s b e f o r e stylar a b s c i s s i o n . T h e r e f o r e , the t i m e pollen t u b e s r e q u i r e to r e a c h the o v a r y is a p p a r e n t l y o f s i g n i f i c a n c e to r e p r o d u c tive efficiency, a n d T T S p r o t e i n s m a y f u n c t i o n to e n s u r e that pollen t u b e s e l o n g a t e at r a t e s a l l o w i n g t h e m to arrive at the o v a r y within the o p t i m a l time for fertilization. T T S p r o t e i n s h a v e to be r e d u c e d to v e r y low levels before a r e d u c t i o n in the p o l l e n t u b e g r o w t h rate or r e d u c e d fertility is m a n i f e s t e d ( F i g u r e s 5 a n d 6). T h i s s u g g e s t s that e i t h e r T T S p r o t e i n s are u s u a l l y in h u g e e x c e s s or that their f u n c t i o n can be s u b s t i t u t e d to c o n s i d e r a b l e e x t e n t s by o t h e r t r a n s m i t t i n g tissue c o n s t i t u e n t s . In a d d i t i o n to T T S proteins, the stylar t r a n s m i t t i n g tissue E C M is e n r i c h e d in m a n y s e c r e t o r y c o m p o u n d s including m a n y g l y c o p r o teins. A l t h o u g h o v e r l a p p i n g f u n c t i o n s from multiple transmitting tissue E C M c o m p o n e n t s to s u p p o r t pollen t u b e g r o w t h r e m a i n s to be d e m o n s t r a t e d , f u n c t i o n a l r e d u n dancy among different molecules and gene redundancy a m o n g the s a m e m o l e c u l e are o f t e n a s s o c i a t e d with proc e s s e s of vital i m p o r t a n c e to an o r g a n i s m to s a f e g u a r d a g a i n s t their failure. M u t a n t plants with s t r u c t u r a l l y and d e v e l o p m e n t a l l y n o r m a l f l o w e r s but a n o m a l o u s pollen t u b e g r o w t h c h a r a c t e r i s t i c s h a v e s o far not b e e n r e c o v e r e d f r o m e x t e n s i v e m u t a n t s c r e e n s in A r a b i d o p s i s . T h u s , isolation and c h a r a c t e r i z a t i o n o f c o m p o n e n t s p r e s e n t a l o n g the pollen t u b e g r o w t h p a t h w a y , direct b i o c h e m i c a l and m o l e c u l a r a n a l y s i s of their i n t e r a c t i o n s with p o l l e n tubes, a n d t r a n s g e n i c i n v e s t i g a t i o n s into their in v i v o f u n c t i o n a l roles are p r o d u c t i v e a p p r o a c h e s in the s t u d y of p o l l e n pistil interactions. Experimental Procedures TTS Protein Purification Styles from prepollinated mature Nicotiana tabacum (W38) flowers were hand-bisected longitudinally to expose the transmitting tissues. They were immersed immediately in TTS protein isolation buffer (84 mM citric acid, 2 mM Na2S204 [pH 3]) at 4°C. This method afforded a 20-fold purification of TTS proteins from total stylar proteins. Besides TTS proteins, other major proteins in this extract were two families of AGPs in the 100-200 kDa and the 65-80 kDa ranges. Proteins were separated on Sephadex G 100 in the TTS protein isolation buffer. Fractions were assayed by immunoblot detection using antibodies directed against the C-terminal nonproline-rich domain of the TTS-1 protein backbone and alkaline phosphatase-conjugated secondary antibodies (Wang et al., 1993). The peak TTS protein-containing fractions were dialyzed into 20 mM Na-phosphate (pH 6), 50 mM NaCI, 2 mM Na=S204 and chromatogrammed on the cationic exchanger carboxymethyI-Sepharose in the same buffer. The residual 100-200 kDa AGPs were removed in the flowthrough. Proteins were eluted by a linear gradient of 0.05 M to 0.5 M NaCI. TTS proteins eluted at around 0.3 M NaCI and were resolved from the 65-80 kDa non-TTS proteins that eluted between 0.1-0.15 M NaCI. Purified TTS proteins were dialyzed into 20 mM Na-phosphate (pH 6), 150 mM NaCI, 2 mM Na2S~O,, and stored at -20°C. About 500 ~g of TTS proteins were recovered from 500 hand-bisected styles in each preparation. TTS protein molecules aggregated easily and irreversibly cross-linked with one another in vitro. The extent of protein aggregation was assessed for each preparation. Chemical Analysis of TTS Proteins Chemical deglycosylation by hydrogen fluoride was as described (van Hoist and Varner, 1984). Amino acid analysis was carried out by The Walton Jones Cell Science Center. Peptide sequencing was carried out by the W. M. Keck Foundation at Yale University. Sugar composi-

tion and linkage analyses (York et al., 1985) were carried out by the Complex Carbohydrate Research Center at the University of Georgia. RNA and Protein Blot Analysis RNA and protein isolation and RNA blot and protein immunoblot analyses were as described (Wang et al., 1993). For detection of AGPs, protein blots were reacted with I~-glucosyl Yariv reagent (0.1 mg/ml) (Biosupplies Australia) as described (Baldwin et al., 1993). Electron Microscopy TTS proteins were dialyzed into water, lyophilized, and resuspended in 50% glycerol, I0 mM KCI at 0,3 mg/ml, They were rotary shadowed (Sommerville and Seheer, 1987) and viewed on a Zeiss EM at 60 kV. In Vitro Pollen Germination and Tube Growth Cultures Pollen (W38) germination and tube growth cultures were carried out in microtiter dishes in a sugar-depleted growth medium (20 mM MES [pH 6], 3 mM Ca(NO3)2.4H20, 1 mM KNO~, 0.8 mM MgSO,.7H20, 1.6 mM H3BO3), or in the same medium supplemented with TTS proteins or various sugars that were added at the time of pollen inoculation. Polyethylene glycol was not included in this growth medium. Germination medium (100 I~1)with 10 pollen grains/pl were used in each assay. Only freshly prepared or once-frozen TTS proteins with little aggregation were used in these assays. When chemically deglycosylated TTS proteins were used, purified native TTS proteins were treated as described above, precipitated by acetone, and resuspended in 20 mM Na-phosphate (pH 6), 50 mM NaCI, 2 mM Na2S204. They were chromatogrammed on Sephadex G50. The 30 kDa deglycosylated TTS protein species was collected, dialyzed into water, lyophized, and resuspended in water before use. Semi-In Vivo Pollen Tube Growth Assays Pistils from W38 flowers at 1 day before anthesis were hand-pollinated with pollen from two freshly dehisced anthers. After 26 hr, pollen tubes began to enter the ovary. The styles were cut with a sharp razor blade at 0.5 cm above the style-ovary junction and cultured on the sugar-free pollen tube growth medium solidified by 0.5% agarose on a microscope slide. The cut styles were immediately sealed with the same medium. They were cultured in a humid chamber at 28°C for about 10 hr. Pollen tubes should begin to emerge from the cut ends at about this time. Pollen tube growth is extremely sensitive to conditions associated with wounding. Pollen tubes never emerged from some styles, or appeared abnormal as they emerged. Only samples with normal pollen tubes between 0.5-1 mm long and elongating parallel to the long axis of the style were used in these assays. A 5 i11 agarose plug made in the same medium as on the slide and containing either TTS proteins or other additives was inserted into a slit made at the pollen tube front and at various distances from the styles. Only freshly prepared or once-frozen TTS proteins were used in these assays. Pollen tube growth was normal for at least 24 hr. The pollen tube growth characteristic was observed after about 20 hr of growth by light microscopy, or by epifluorescence microscopy after aniline blue staining (Jefferies and Belcher, 1974). A gradient of increasing TTS protein concentrations established by diffusion from the source was confirmed by protein blot analysis of the proteins recovered from the surrounding agarose (data not shown). The time and position of agarose plug implantation relative to the position of the pollen tube front, the rate of TTS protein diffusion, and the rate of pollen tube growth were all critical parameters in these assays. Plant Transformation and the Analysis of Fertility Phenotype N, tabacum (W38 or SR1) grown in tissue culture conditions were used in Agrobacterium-mediated transformation (Delebrese et al., 1986). One of the constructs, 35STTS, has a chimeric gene with a double CaMV 35S promoter fused to a full length TTS-2 cDNA (Cheung et al., 1993). The second construct, anti-TTS, has a chimeric TTS-1 gene promoter (X.-y. Zhan, H.-m. W., and A. Y. C., unpublished data) fused to antisense TTS-2 cDNA. The reproduction-related phenotype was examined in detail only in plants with TTS protein amounts suppressed by these transgenes to less than 25% of the level found in control plants. The fertility phenotype in these transgenic plants was assessed by comparing the fruit capsule sizes and their seed contents with those

Cell 392

in control plants after natural self-pollination. Detailed analysis were carried out for the transgenic plants with highly reduced TTS protein levels by two different methods. Flowers at 1 day prior to anthesis were emasculated and pollinated with two anthers full of pollen grains. For direct observation of pollen tube growth, pollinated pistils were excised at 24 hr or at 30 hr (for W38 or SR1 derivatives, respectively) after pollination when pollen tubes reached the bottom of the styles. The styles were cut into 0.5 cm segments from the stigma to the bottom of the style, fixed and stained with aniline blue, and observed by epifluorescence microscopy. Fertility was also assessed based on seed yields after interrupted pollination. In these assays, pollinated styles were cut at 0.5 cm above the style-ovary junction at 26 hr or 28 hr, or at 31 hr or 33 hr (for W38 or SR1 derivatives, respectively) after pollination. For "uninterrupted" pollen tube growth, styles were cut at 0.5 cm above the style-ovary junction at 72 hr after pollination. Fruits were removed at 1 month after pollination when all the seeds have fully dessicated. Seed sets were the better indicator of reproductive yields because differences in fruit capsule sizes were often smaller than differences in seed yields.

Acknowledgments We thank Mr. L. Sablitz and D. Garinger for maintaining tobacco plants, Mr. B. Piekos for advice and assistance on electron microscopy, and Mrs. N. Carrignan for assistance in the preparation of this manuscript. We thank Drs. H. Dickinson, E. Lord, J. Mascarenhas, S. Morga~n, M. Mooseker, T. Nelson, N. Schultes, and J. Varner for suggestions and questions on an earlier version of this manuscript that helped to clarify the concepts we present. We also thank Dr. J. P. Trinkaus for encouragement. We are most grateful to Dr. J. Haley for her generous help and insightful suggestions on this manuscript. H. W. was supported by the Rockefeller Foundation. This work was partially supported by a grant from the U nited States Department of Agriculture and a Department of Energy grant to the Center for Plant and Microbial Complex Carbohydrates.

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Received April 25, 1995; revised June 9, 1995.

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