Wang et al. — A desiccation-related protein in lily pollen

Bot. Bull. Acad. Sin. (1999) 40: 199-205

A stress-inducible protein associated with desiccation in lily pollen

C.-S. Wang¹, S.-M. Lin, and S.-L. Wei

Graduate Institute of Agricultural Biotechnology, National Chung Hsing University, Taichung, Taiwan, Republic of China

(Received June 11, 1998; Accepted November 24, 1998)

Abstract. This work characterizes a stress-inducible protein (LLA) associated with desiccation in lily (Lilium longiflorum Thunb. cv Snow Queen) pollen. Immunoblots of two-dimensional polyacrylamide gels of pollen protein indicated that the LLA-32 protein with a molecular mass of 32 kDa was heterogeneous and had an isoelectric point of 5.6. The protein was developmentally regulated at the later stage of pollen maturation during anther development. The accumulation of LLA-32 proteins remained steady in the mature pollen, but the protein disappeared during pollen germination. Subcellular fractionation of pollen proteins revealed that the protein was detected only in the cytoplasmic fraction, indicative of a gametophytic origin. Premature drying of developing pollen demonstrated that the LLA-32 doublet was associated with desiccation. The accumulation of the proteins can be experimentally manipulated by methyl jasmonate and salicylic acid as well as by mannitol and methyl viologen. Differential accumulation of the doublet polypeptides occurred with response to various stimuli. The upper band of LLA-32 doublet polypeptides markedly accumulated upon dehydration whereas the lower band of LLA-32 doublet significantly increased its level of accumulation when other stresses were applied.

Keywords: Desiccation; Lilium longiflorum; Pollen protein; Stress-inducible.

Introduction

Anthers are floral structures specialized for pollen production in higher plants. In young anthers, the sporogenous cells undergo meiosis and give rise to the formation of microspores at the tetrad stage. These microspores develop into microgametophytes after haploid mitosis and subsequently differentiate into mature pollen (Johri, 1984). In the final stage of maturation, pollen exhibits various degrees of desiccation and this natural drying process promotes pollen germinability to a great extent.

As an adaptive organ that facilitates the colonization of terrestrial habitats, pollen develops a number of unique features (Blackmore and Knox, 1990). First, the pollen grain is surrounded by a protective wall consisting of two layers, exine and intine. Second, sporopollenin in the exine is an insoluble polymer that is extremely resistant to drought and chemicals, including strong acids. Finally, the presence of soluble sugars such as sucrose in pollen may play an essential role in the acquired tolerance to desiccation (Hoekstra and van Roekel, 1988). Anhydrobiotic organisms are known to contain trehalose, a nonreducing disaccharide of glucose that is related to acquired toler

ance to severe dehydration (Crowe et al., 1984). In addition to sugars, proteins are considered to be another component of desiccation tolerance. Various types of drought-inducible proteins have been identified (Skriver and Mundy, 1990; Shinozaki and Yamaguchi-Shinozaki, 1997). Among these, dehydrins are known to accumulate immediately before desiccation during seed development (Close et al., 1989; Dure, 1993). Dehydrins are also reported in mature pollen grains (Michel et al., 1994; Wang and Cutler, 1995). It has been suggested that these proteins play a role in protecting plant structures during water loss. Most genes that respond to drought are also abscisic acid (ABA)-responsive (Ingram and Bartels, 1996; Bray, 1997). We recently described two pollen-specific proteins that were immunologically unrelated to dehydrins in lily seeds (Wang et al., 1996, 1998). The two pollen proteins that accumulated shortly before anthesis were ABA and polyethylene glycol (PEG)-inducible (Wang et al., 1996). Herein, we continue our examination by using immunological and biochemical methods to characterize one of the two proteins during development and stress. The LLA-32 protein is a heterogeneous protein located in the cytoplasmic fraction of pollen grains. Premature drying of developing pollen demonstrates that the doublet polypeptides are associated with desiccation. The LLA-32 doublet polypeptides differentially increase their levels of accumulation upon dehydration and other stresses.

¹Corresponding author. Tel: 886-4-287-4754 ext. 105, 106; Fax: 886-4-286-1905; E-mail: cswang2@dragon.nchu.edu.tw

Botanical Bulletin of Academia Sinica, Vol. 40, 1999

Materials and Methods

Plant Materials

Plants of lily (Lilium longiflorum Thunb. cv Snow Queen) were grown in the field. Lily buds ranging from 7 to 175 mm were harvested at autumn and dissected to isolate anthers. Pollen was manually collected before anthesis on the basis of bud length or at anthesis (from 170 to 175-mm buds) or at indicated days after anthesis (DAA). Microspore mitosis was complete at a bud length around 65 mm, and pollen subsequently entered the maturation phase of development. To assure that the growing plants were healthy and free of pathogenic attack, the normal patterns of desiccation-associated proteins that accumulated in the developing anthers were monitored. Unless indicated elsewhere, pollen collected 1 day before flowering was used for germination experiments. Material was stored at -80°C until use.

Premature Drying and Germination Treatments

Pollen taken from anthers of different flower bud sizes was air-dried on the laboratory bench for 24 h at 25°C. For pollen germination, pollen (approximately 300 mg) was incubated in 4 ml of germination medium [0.29 M pentaerythritol, 300 µg ml^-1 Ca(NO₃)₂•4H₂O, 10 µg ml^-1 H₃BO₃ and 100 µg ml^-1 KNO₃] (Dickinson, 1978) at 30°C with shaking for 24 h. Where indicated, pollen was incubated in the medium containing various concentrations of salicylic acid (SA, sodium salt, Sigma), methyl jasmonate (MeJA, Bedoukian Research Inc., Danbury, CT, USA), mannitol or methyl viologen. To avoid imbibitional damage, frozen or dried pollen was routinely placed in humid air for 1 h prior to testing for germination (Hoekstra, 1984).

Preparation, Electrophoresis and Immunoblotting of Pollen Protein

The phenol extraction method was used to extract total protein from lily pollen (Wang et al., 1992). The total pollen protein was then subjected to two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The extraction of heat-stable proteins from pollen was performed according to Wang et al. (1996). Protein concentration was determined by the dye binding Bio-Rad protein assay according to the supplier's directions. The heat-stable protein fractionated by SDS-PAGE was overrun in order to have a better separation of the LLA-32 doublet polypeptides. The heat-stable protein on the gel was either stained with silver, or electroblotted onto nitrocellulose (0.45 µm, Gelman Sciences, Ann Arbor, MI, USA) (Wang et al., 1996). The 2D-PAGE-purified LLA-32 polypeptides (approximately 10 µg) in gel slices were ground in 0.5 ml of PBS (10 mM sodium phosphate, pH 7.4, 150 mM NaCl) and emulsified with 0.5 ml Freund's complete adjuvant (Sigma). The emulsion was intramuscularly injected into a local hen L2-BDS (kindly provided by Dr. Y. P. Lee, Dept. of Animal Science, National Chung Hsing

Univ., Taichung, Taiwan). The second injection with approximately 10 µg protein in PBS/Freund's incomplete adjuvant was administered 4 weeks after the first one. A booster injection with the same amount of protein in PBS/Freund's incomplete adjuvant was then administered 1 week after the second injection. Eggs were collected a week after the booster injection. Immunoglobulins (Igs) were isolated from the egg yolks, aliquoted, and stored at -80°C in the presence of 0.1% sodium azide. Pre-immune IgGs used as controls were taken from eggs before injection. For each preparation of blot-affinity-purified antibodies, 750 µl of egg yolk IgG was used according to the method of Smith and Fisher (1984). Antibodies were eluted with elution buffer (5 mM glycine-HCl, pH 2.3, 500 mM NaCl, 0.5% [v/v] Tween 20, and 100 µg ml^-1 BSA). The eluates were immediately neutralized by the addition of 1 M sodium phosphate (pH 7.7) to a final concentration of 50 mM. The eluates were aliquoted and stored at -80°C. Blots were immunostained using a 1:4 dilution of eluate of the affinity-purified LLA-32 antibodies.

Subcellular Fractionation of Pollen Proteins

Mature pollen (5 g) was suspended in 10 ml of 0.1 M Tris-HCl pH 8.0 containing 0.29 M pentaerythritol and 2.4 mM PMSF. Pollen was shaken for 1~2 min and centrifuged at 5,000 g for 3 min (water soluble fraction in the pollen wall). This process was repeated three times. Pollen remained intact under the above treatment when examined by microscopy. The final washed pollen was disrupted by passing it twice through a French Press (SLM-AMINCO FA-078, Rochester, NY, USA) at 10,000 p.s.i. in the Tris buffer as described above. The broken cells were centrifuged at 12,000 g to produce a crude wall fraction (pellet) and a cytoplasmic fraction (supernatant) (Chay et al., 1992). The pellet was successively washed in the following ways, each followed by centrifugation: four times with 100 ml of 0.05 M Tris-HCl pH 8.0, 12~15 times with 100 ml of deionized distilled water, once with 50 ml of 1% Triton X-100, once with 100 ml of deionized distilled water, once with 50 ml of 1% Triton X-100, and 8~11 times with deionized distilled water (Li et al., 1983). All washings were done at 4°C. The following extraction of protein from each fraction was according to the phenol method (Wang et al., 1992).

Results

LLA-32 Protein is Heterogeneous and Developmentally Regulated

Total protein extracted from mature pollen was fractionated by 2D-PAGE. A 2D-PAGE immunoblot was incubated with affinity-purified anti-LLA-32 antibodies to show that the LLA-32 protein was heterogeneous and acidic, having an isoelectric point (pI) of 5.6 (Figure 1A). Affinity-purified antibodies to LLA-32 recognized three polypeptides, one with a slightly higher molecular weight (Figure 1B). 2D-PAGE of total pollen protein revealed that

Wang et al. — A desiccation-related protein in lily pollen

Figure 2. Accumulation of LLA-32 proteins in developing anthers of L. longiflorum. Heat-stable proteins were extracted from various size classes of anthers: 1 = 9 mm (16-mm buds); 2 = 17 mm (35-mm buds); 3 = 21 mm (70-mm buds); 4 = 22 mm (90-mm buds); 5 = 22 mm (110-mm buds); 6 = 22 mm (130-mm buds); 7 = 23 mm (140-mm buds); 8 = 23 mm (150-mm buds); 9 = 23 mm (160-mm buds); 10 = 23 mm (from 170 to 175-mm buds, at anthesis) and from mature pollen at indicated days after anthesis (DAA): 11 = 1 DAA; 12 = 3 DAA; 13 = 5 DAA. Total protein was extracted from each anther size classes and from mature pollen and heat-treated at 90°C for 10 min. Heat-stable proteins (5 µg) were fractionated by SDS-PAGE, electroblotted onto nitrocellulose, and immunochemically detected using affinity-purified LLA-32 antibodies.

Figure 1. The heterogeneity of pollen LLA-32 proteins in L. longiflorum. Total protein (120 µg) extracted from pollen was electrophoresed by 2D-PAGE and either stained with silver (A) or electroblotted onto nitrocellulose, and immunologically detected using affinity-purified LLA-32 antibodies (B). Instead of using 120 µg, 2.4 mg of total protein was fractionated and electroblotted for the immunodetection of LLA-32 proteins. The LLA-32 proteins are indicated by arrows. Positions of molecular mass marker proteins are indicated.

antibodies to LLA-32 only specifically recognized LLA-32 proteins. No other protein was detected by affinity-purified LLA-32 antibodies. Therefore, instead of using total protein, heat-stable proteins were extracted and used in the following experiments to simplify the protein patterns in a gel.

To determine whether LLA-32 protein could be detected in the phases of premeiotic and microspore development, heat-stable proteins from various size classes of anthers were fractionated by SDS-PAGE. An immunoblot revealed that LLA-32 was detected only at the later stage of pollen maturation (Figure 2). The temporal distribution of LLA-32 during anther development was consistent with the earlier report of its detection in the phase of pollen maturation by silver staining (Wang et al., 1992). In addition, the accumulation of LLA-32 proteins in the mature pollen remained steady even after five DAA. However, the proteins disappeared when pollen was incubated in the germinating buffer for 4 h or longer (Figure 3). The germinating buffer contains 0.29 M pentaerythritol instead of 0.3 M sucrose.

Subcellular Fractionation of LLA-32 Proteins

To investigate the subcellular location of LLA-32 protein, pollen was initially suspended in Tris buffer pH 8.0 and shaken to release water-soluble proteins that were

Figure 3. Time course of the disappearance of LLA-32 proteins in germinating pollen of L. longiflorum. Mature pollen (0.2 g) was incubated at 30°C in the germination medium at indicated hours after germination. Total protein was extracted from ungerminated and germinated pollen and heat-treated at 90°C for 10 min. Heat-stable proteins (5 µg) were fractionated by SDS-PAGE, electroblotted onto nitrocellulose, and immunochemically detected using affinity-purified LLA-32 antibodies (A). The LLA-32 protein levels (B), estimated by scanning the optical density of immunostained signals shown in (A), are expressed as percentages of the maximal levels found in ungerminated pollen. Each data point is the average of signals from three independent immunoblots.

Botanical Bulletin of Academia Sinica, Vol. 40, 1999

embedded in the pollen wall. The final washed pollen was disrupted through a French Press, and then the broken cells were centrifuged to produce a crude wall/membrane fraction (pellet) and a cytoplasmic fraction (supernatant). Next, the pellet was successively washed with buffers (in Materials and Methods) and no protein in the supernatant was detected by Bio-Rad protein assay after the final washing with deionized distilled water (data not shown). Pollen protein extracted from each fraction was resolved by SDS-PAGE (Figure 4A). Immunoblots of total protein from each fraction revealed that the LLA-32 proteins were undetected in the pollen wall (lanes B1 and B3). The proteins were detected only in the cytoplasmic fraction, indicative of a gametophytic origin (lanes B2).

Influence of Premature Drying on the Accumulation of LLA-32 Proteins

To determine drying effect on the accumulation of LLA-32 proteins, pollen was removed from developing anthers of indicated bud lengths and air-dried for 24 h. Heat-stable proteins were extracted from various developmental stages of pollen from 140 to 170-mm buds and from mature pollen at anthesis (from 170 to 175-mm buds). SDS-PAGE analysis indicated that a number of heat-stable polypeptides, including the LLA-32 proteins, increased their levels of accumulation upon premature drying (Figure 5A). Immunoblot analyses also revealed that the upper band of LLA-32 doublet significantly increased its level of accumulation whereas no appreciable induction of the lower band of LLA-32 doublet occurred upon drying (Figure 5B).

Influence of Various Stresses on the Accumulation of LLA-32 Proteins

As reported elsewhere, some dehydration-responsive genes are also induced by osmotic stress in various plant tissues (Leonardi et al., 1995). The LLA-32 proteins are both PEG-8000 and ABA-inducible (Wang et al., 1996). To further investigate the inductive effect of various environmental stresses on the LLA-32 proteins, we took advantage of the disappearance of the proteins during pollen germination (Figure 6, lane 2). After incubation with various stimuli in germinating buffer for 24 h, pollen protein was extracted, heat-treated, and fractionated by SDS-PAGE. Immunoblot analyses revealed that a maximum accumulation of LLA-32 proteins was observed when 180 µM MeJA, 1% mannitol, 30 µM SA, or 1 mM methyl viologen was applied (Figure 6). Contradictory to a marked induction of the upper band of LLA-32 proteins upon dehydration, the lower band of LLA-32 proteins significantly increased its level of accumulation when other stresses were applied. However, the protein levels dropped dramatically when the concentration reached 10% mannitol or 100 µM SA whereas the protein levels remained steady when the concentration of MeJA or methyl viologen increased to 540 µM or 10 mM, respectively (data not shown).

Figure 4. Subcellular immunodetection of LLA-32 proteins in the mature pollen of L. longiflorum. Total protein (2 µg) from each fraction was electrophoresed by SDS-PAGE and either stained with silver (A) or electroblotted onto nitrocellulose, and immunochemically detected using affinity-purified LLA-32 antibodies (B). Instead of using 2 µg, 6 µg of heat-stable proteins was fractionated and electroblotted for immunochemical detection. Water-soluble fraction of pollen wall (lane 1), cytoplasmic fraction of pollen grains (lane 2), and pollen wall/membrane fraction (lane 3). Positions of molecular mass marker proteins are indicated.

Figure 5. Effect of premature drying on the accumulation of LLA-32 in developing pollen of L. longiflorum. Pollen was removed from anthers of three consecutive flower bud sizes: 140~150 mm (lane 1), 150~160 mm (lane 2), 160~170 mm (lane 3) or at anthesis (lane 4). Pollen protein was extracted and heat-treated at 90°C for 10 min. Heat-stable protein (2 µg) was fractionated by SDS-PAGE and either silver-stained (A) or electroblotted onto nitrocellulose, and immunochemically detected using affinity-purified LLA-32 antibodies (B). Instead of using 2 µg, 6 µg of heat-stable proteins was fractionated and electroblotted for immunochemical detection. Positions of molecular mass marker proteins are indicated.

Wang et al. — A desiccation-related protein in lily pollen

Discussion

In our previous report on the proteins of lily anthers, we suggested that the LLA-32 protein is pollen-specific (Wang et al., 1996). In this study, affinity-purified antibodies were used to determine the heterogeneity and distribution of the protein during pollen development and germination. 2D-PAGE immunoblot resolved the doublet of LLA-32 into a number of polypeptides. Subcellular fractionation of pollen proteins indicated that LLA-32 proteins were not in the pollen wall. Thus, the protein does not belong to the family of many sporophytic proteins in the pollen wall involved in cell-cell recognition (Heslop-Harrison and Heslop-Harrison, 1973). The protein was observed in the cytoplasmic fraction of pollen grains (Figure 4), implying a gametophytic nature. Because proteins in the intine layer of the pollen wall may be released to the cytoplasmic fraction upon the breakage of pollen grains, the location of LLA-32 proteins in the intine layer can not be excluded.

The accumulation of LLA-32 polypeptides in anther development correlates with desiccation that occurs naturally in the pollen (Wang et al., 1996). Premature drying treatment of developing pollen further demonstrates that the accumulation of the proteins is associated with desiccation (Figure 5). This finding corresponds to the observation that proteins in dry pollen are resistant to drying and freeze-drying (Wolkers and Hoekstra, 1995). As widely suggested, dehydrins act as an essential component against water stress; however, they may need to work in concert with oligosaccharides to develop desiccation tolerance (Blackman et al., 1992). The LLA-32 proteins, which are not dehydrins (Wang et al., 1996), may belong to a novel class of proteins related to dehydration. It is interesting that one of the doublet polypeptides responds to dehydration more significantly than the other (Figure 5).

Kermode (1990) contended that dehydration in developing seeds is a critical switch, from a developmental to a germination program. The developmental switch is acquired at seed ages closer to full maturity, during which desiccation occurs and specific proteins accumulate. Developing pollen exhibits various degrees of desiccation immediately before maturation. Therefore, a similar switch from a developmental to a germination mode may exist in pollen. Premature drying treatment of developing pollen possibly results in a suppressive developmental program and a "switching-on" of the germination program. In that sense, the appearance of LLA-32 proteins may serve as an indicator for such a switch.

In addition to being induced by dehydration, the LLA-32 proteins are MeJA-inducible (Figure 6A). Similar to the LLA-32 proteins, several jasmonate-induced proteins (JIPs), which accumulate under water stress conditions, have been reported elsewhere (Reinbothe et al., 1992, 1994). The LLA-32 protein is also ABA-inducible (Wang et al., 1996). Plant cells generally react to various environmental stresses in different ways, but the production of ABA-responsive (Rab) proteins is shared by most all

Figure 6. Induction of LLA-32 proteins in pollen of L. longiflorum by various stimuli during germination. Pollen harvested 1 day before anthesis (lane 1) was incubated in a germination buffer at 30°C for 24 h without any stimulus (lane 2), or with the application of 45, 90 and 180 µM MeJA (lanes A3-5); of 0.1, 0.5 and 1% mannitol (lanes B3-5); of 1, 10, and 30 µM SA (lanes C3-5) and of 10, 100 and 1000 µM methyl viologen (lanes D3-5). Pollen protein was extracted and heat-treated at 90°C for 10 min. Heat-stable proteins (6 µg) were fractionated by SDS-PAGE, electroblotted onto nitrocellulose, and immunochemically detected using affinity-purified LLA-32 antibodies, respectively.

of them (Leonardi et al., 1995). It has been proposed that water loss induces an increase in endogenous ABA level that subsequently activates JA biosynthesis, resulting in an increase of endogenous JA. Correspondingly, an increase in JA level activates the expression of wound-inducible genes (Wasternack and Parthier, 1997). This model might account for why many of those JIPs are also ABA-inducible (Reinbothe et al., 1994). Nevertheless, some identified JIPs do not exhibit an induction by ABA (Moons et al., 1997; Wasternack and Parthier, 1997).

The LLA-32 proteins are also induced by PEG (Wang et al., 1996) and mannitol (Figure 6B). In addition, the LLA-32 proteins are methyl viologen-inducible (Figure 6D). Methyl viologen, a potent oxidant, is known to enhance O₂^_ production that may be involved in the initiation of oxidative damage (Iturbe-Ormaetxe et al., 1998). Moreover, the LLA-32 proteins are SA-inducible (Figure 6C). SA is

Botanical Bulletin of Academia Sinica, Vol. 40, 1999

of Papaver dubium L. pollen during its development in the anther: possible role of phospholipid composition and sucrose content. Plant Physiol. 88: 626-632.

Ingram, J. and D. Bartels. 1996. The molecular basis of dehydration tolerance in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47: 377-403.

Iturbe-Ormaetxe, I., P.R. Escuredo, C. Arrese-Igor, and M. Becana. 1998. Oxidative damage in pea plants exposed to water deficit or paraquat. Plant Physiol. 116: 173-181.

Johri, B.M. (ed.). 1984. Embryology of Angiosperms. Springer-Verlag, Berlin, Heidelberg, New York, Tokyo, 197 pp.

Kermode, A.R. 1990. Regulatory mechanisms involved in the transition from seed development to germination. Cri. Rev. Plant Sci. 9: 155-195.

Leonardi, A., S. Heimovaara-Dijkstra, and M. Wang. 1995. Differential involvement of abscisic acid in dehydration and osmotic stress in rice cell suspension. Physiol. Plant. 93: 31-37.

Li, Y.-Q., A.F. Croes, and H.F. Linskens. 1983. Cell-wall proteins in pollen and roots of Lilium longiflorum: extraction and partial characterization. Planta 158: 422-427.

Michel, D., A. Furini, F. Salamini, and D. Bartel. 1994. Structure and regulation of an ABA- and desiccation-responsive gene from the resurrection plant Craterostigma plantagineum. Plant Mol. Biol. 24: 549-560.

Moons, A., E. Prinsen, G. Bauw, and M. van Montagu. 1997. Antagonistic effects of abscisic acid and jasmonates on salt stress-inducible transcripts in rice roots. Plant Cell 9: 2243-2259.

Reinbothe, S., B. Mollenhauer, and C. Reinbothe. 1994. Jips and rips: The regulation of plant gene expression by jasmonates in response to environmental cues and pathogens. Plant Cell 6: 1197-1209.

Reinbothe, S., C. Reinbothe, J. Lehmann, and B. Parthier. 1992. Differential accumulation of methyl jasmonate-induced mRNAs in response to abscisic acid and desiccation in barley (Hordeum vulgare). Physiol. Plant. 86: 49-56.

Ryals, J., S. Uknes, and E. Ward. 1994. Systemic acquired resistance. Plant Physiol. 104: 1109-1112.

Shinozaki, K. and K. Yamaguchi-Shinozaki. 1997. Gene expression and signal transduction in water-stress response. Plant Physiol. 115: 327-334.

Skriver, K. and J. Mundy. 1990. Gene expression in response to abscisic acid and osmotic stress. Plant Cell 2: 503-512.

Smith, D.E. and P.A. Fisher. 1984. Identification, developmental regulation, and response to heat shock of two antigenetically related forms of a major nuclear envelope protein in Drosophila embryos: application of an improved method for affinity purification of antibodies using polypeptides immobilized on nitrocellulose blots. J. Cell Biol. 99: 20-28.

Wang, C.-S., Y.-E. Liau, J.-C. Huang, T.-D. Wu, C.-C. Su, and C.H. Lin. 1998. Characterization of a desiccation-related protein in lily pollen during development and stress. Plant Cell Physiol. 39: 1307-1314.

Wang, C.-S., L.L. Walling, K.J. Eckard, and E.M. Lord. 1992. Patterns of protein accumulation in developing anthers of Lilium longiflorum correlate with histological events. Am. J. Bot. 79: 118-127.

Wang, C.-S., T.-D. Wu, C.-K.W. Chung, and E.M. Lord. 1996. Two classes of pollen-specific, heat-stable proteins in Lilium longiflorum. Physiol. Plant. 97: 643-650.

another endogenous signal that apparently plays a prominent role in defense against pathogenic attack, particularly in a localized tissue death where pathogen penetrates (Yang et al., 1997). Besides, SA is a signal in the pathway leading to systemic acquired resistance (SAR) (Ryals et al., 1994). The fact that the LLA-32 proteins can be induced by SA implies that these desiccation-related proteins may also be pathogen-induced. We have detected significant increases of LLA-32 proteins even at the earlier stages of pollen maturation during which the protein is normally undetectable; in this case, the batch of growing lily plants from which anthers were dissected to isolate proteins has been attacked by unknown pathogens (data not shown).

Herein, we report on a heterogeneous, desiccation-related protein that developmentally accumulates immediately before anthesis during anther/pollen development. The differential accumulation of the proteins can be experimentally manipulated by premature drying, plant growth regulators, or other stresses.

Acknowledgments. We wish to thank Mr. C.-J. Chen and his wife for growing lily plants in the field. We also thank Y.-E. Liau, J.-C. Huang and Y.-Y. Yang for collecting pollen and their able technical assistance. This work was supported by National Science Council Grant NSC87-2311-B005-033 to C.-S. Wang.

Literature Cited

Blackman, S.A., R.L. Obendorf, and A.C. Leopold. 1992. Maturation proteins and sugars in desiccation tolerance of developing soybean seeds. Plant Physiol. 100: 225-230.

Blackmore, S. and R.B. Knox (eds.). 1990. Microspores: Evolution and Ontogeny. Academic Press, San Diego, CA., 1 pp.

Bray, E.A. 1997. Plant responses to water deficit. Trends Plant Sci. 2: 48-54.

Chay, C.H., E.G. Buehler, J.M. Thorn, T.M. Whelan, and P.A. Bedinger. 1992. Purification of maize pollen exines and analysis of associated proteins. Plant Physiol. 100: 756-761.

Close, T.J., A.A. Kortt, and P.M. Chandler. 1989. A cDNA-based comparison of dehydration-induced proteins (dehydrins) in barley and corn. Plant Mol. Biol. 13: 95-108.

Crowe, J.H., L.M. Crowe, and D. Chapman. 1984. Preservation of membranes in anhydrobiotic organisms: the role of trehalose. Science 223: 701-703.

Dickinson, D.B. 1978. Influence of borate and pentaerythritol concentrations on germination and tube growth of Lilium longiflorum pollen. J. Am. Soc. Horti. Sci. 103: 413-416.

Dure, L. 1993. Structural motifs in lea proteins. In T.J. Close and E.A. Bray (eds.), Plant Responses to Cellular Dehydration During Environmental Stress. Current Topics in Plant Physiology, vol. 10. An American Society of Plant Physiologists Series, Rockville, Maryland, pp. 91-104.

Heslop-Harrison, J. and Y. Heslop-Harrison. 1973. Pollen-wall proteins: `gametophytic' and `sporophytic' fractions in the pollen walls of the malvaceae. Ann. Bot. 37: 403-412.

Hoekstra, F.A. 1984. Imbibitional chilling injury in pollen: involvement of the respiratory chain. Plant Physiol. 74: 815-821.

Hoekstra, F.A. and T. van Roekel. 1988. Desiccation tolerance

Wang et al. — A desiccation-related protein in lily pollen

Wang, H. and A.J. Cutler. 1995. Promoters from kin1 and cor6.6, two Arabidopsis thaliana low-temperature- and ABA-inducible genes, direct strong beta-glucuronidase expression in guard cells, pollen and young developing seeds. Plant Mol. Biol. 28: 619-634.

Wasternack, C. and B. Parthier. 1997. Jasmonate-signalled plant gene expression. Ternds Plant Sci. 2: 302-307.

Wolkers, W.F. and F.A. Hoekstra. 1995. Aging of dry desiccation-tolerant pollen does not affect protein secondary structure. Plant Physiol. 109: 907-915.

Yang, Y., J. Shah, and D.F. Klessig. 1997. Signal perception and transduction in plant defense responses. Gene Devel. 11: 1621-1639.

¦Ê¦Xªá¯»¤@»P°®À꦳Ãö¤§°f¹Ò»¤µo³J¥Õ

¤ý°ê²» ªL²Q¬ü ÃQ§µ­Û

°ê¥ß¤¤¿³¤j¾Ç¹A·~¥Íª«¬ì§Þ¾Ç¬ã¨s©Ò

¥»¹êÅç¤ÀªR¦Ê¦X (Lilium longiflorum Thunb. cv. ³·¦Z) ªá¯»¤¤¤@»P°®Àꥢ¤ô¦³Ãöªº°f¹Ò»¤µo³J¥Õ (LLA)¡Cªá¯»³J¥Õ¤§Âù¦V¹qªa¤Î§K¬ÌÂàº{¤ÀªRÅã¥Ü LLA-32 ¬O¨ã¦³¦h«¬¡Aµ¥¹qÂI¬° 5.6¡A¤À¤l¶q¬° 32 kDa ªº¦hÐ`¤Ó¡C¦¹ªá¯»³J¥Õ¦bªáÃĵo¨|ªº¹Lµ{¤¤¡A¥u¦bªá¯»¦¨¼ôªº±ß´Á¤~²Ö¿nªí²{¡Cªá¯»¦¨¼ô¥H«á LLA-32 ³J¥Õªº¶q¤´ºû«ù¤@©w¡A¦ý¬O·íªá¯»µÞµo¥|¤p®É«á¡A LLA-32 ³J¥Õ§Y®ø¥¢¡Cªá¯»³J¥Õ¸g¦¸²Ó­M¤ÀÂ÷ªº¹êÅçÅã¥Ü LLA-32 ³J¥Õ¬O¦ì©óªá¯»ªº²Ó­M½è¤º¡C±Nµo¨|¤¤ªºªá¯»¥ý¦æ°®Àê³B²z¥iµý©ú¦¹³J¥Õ½T»P°®À꦳Ãö¡C LLA-32 doublet ³J¥Õªº²Ö¿n¥i¸g¥Ñ¥~¥[ methyl jasmonate¡Bsalicyilic acid¡Bmannitol ©M methyl viologen µ¥¤è¦¡¨Ó¥[¥H ¾Þ±±¡CLLA-32 doublet ¨ü°®Àê©M¨ä¥L°f¹Ò¦Ó¦³¤£¦Pµ{«×ªº²Ö¿n¡C LLA-32 doublet ªº¤@­Ó¦hÐ`¤Ó¨ü°®Àê¼vÅT¦Ó ©úÅã¼W¥[¡FLLA-32 doublet ªº¥t¤@­Ó¦hÐ`¤Ó«h¨ü¨ä¥L°f¹Òªº¼vÅT©úÅã¼W¥[ ¡C

ÃöÁäµü¡G °®Àê¡F¦Ê¦X¡Fªá¯»³J¥Õ¡F°f¹Ò»¤µo¡C