Pollen tube growth and guidance: roles of small, secreted proteins

BACKGROUND: Pollination is a crucial step in angiosperm (flowering plant) reproduction. Highly orchestrated pollen-pistil interactions and signalling events enable plant species to avoid inbreeding and outcrossing as a species-specific barrier. In compatible pollination, pollen tubes carrying two sperm cells grow through the pistil transmitting tract and are precisely guided to the ovules, discharging the sperm cells to the embryo sac for fertilization. SCOPE: In Lilium longiflorum pollination, growing pollen tubes utilize two critical mechanisms, adhesion and chemotropism, for directional growth to the ovules. Among several molecular factors discovered in the past decade, two small, secreted cysteine-rich proteins have been shown to play major roles in pollen tube adhesion and reorientation bioassays: stigma/style cysteine-rich adhesin (SCA, approx. 9·3 kDa) and chemocyanin (approx. 9·8 kDa). SCA, a lipid transfer protein (LTP) secreted from the stylar transmitting tract epidermis, functions in lily pollen tube tip growth as well as in forming the adhesive pectin matrix at the growing pollen tube wall back from the tip. Lily chemocyanin is a plantacyanin family member and acts as a directional cue for reorienting pollen tubes. Recent consecutive studies revealed that Arabidopsis thaliana homologues for SCA and chemocyanin play pivotal roles in tip polarity and directionality of pollen tube growth, respectively. This review outlines the biological roles of various secreted proteins in angiosperm pollination, focusing on plant LTPs and chemocyanin.     

Development and Pollination Regulated Accumulation and Glycosylation of a Stylar Transmitting Tissue-Specific Proline-Rich Protein

The extracellular matrix of stylar transmitting tissues of many angiosperms is enriched in secretory materials that are believed to be important for interactions with pollen tubes. We have previously characterized two related cDNAs (TTS-1 and TTS-2) for stylar transmitting tissue-specific proline-rich proteins (TTS proteins) from Nicotiana tabacum. We show here that TTS proteins are highly glycosylated proteins with apparent molecular masses ranging between 50 and 100 kD. Results from chemical and enzymatic deglycosylation suggest that TTS proteins have N-linked glycosyl groups, and the extensive glycosylation most probably has resulted from modifications at the proline residues. TTS proteins are localized to the intercellular regions between neighboring transmitting tissue cells, the space in which pollen tubes elongate as they migrate from the stigma toward the ovary. TTS mRNA and protein levels are regulated during pistil development and by pollination. The levels of TTS mRNAs and proteins increase with flower development and reach the maximal levels as flowers approach anthesis. These maximal levels are maintained in the styles for at least 3 to 4 days after pollination, during which time pollen tubes elongate and reach the ovary. Spatially, TTS mRNAs and proteins accumulate first in the stigmatic end of young styles, and their levels progressively increase toward the basal end as pistils mature. Pollination stimulates the levels of TTS mRNAs and proteins in hand-pollinated young styles, which normally accumulate relatively low levels of these TTS gene products. Pollination also qualitatively affects TTS mRNAs and proteins. In pollinated styles, TTS mRNAs are shorter than those in unpollinated styles and underglycosylated TTS protein species begin to accumulate. The elaborate regulatory mechanisms governing TTS mRNAs and proteins during development and by pollination strongly suggest that these proteins may play a functional role in the process of pollination.     

Evaluation of pollen tube growth ability in Petunia species having different style lengths

Pollen tube growth is essential for the fertilization process in angiosperms. When pollen grains arrive on the stigma, they germinate, and the pollen tubes elongate through the styles of the pistils to deliver sperm cells into the ovules to produce the seeds. The relationship between the growth rate and style length remains unclear. In previous studies, we developed a liquid pollen germination medium for observing pollen tube growth. In this study, using this medium, we examined the pollen tube growth ability in Petunia axillaris subsp. axillaris, P. axillaris subsp. parodii, P. integrifolia, and P. occidentalis, which have different style lengths. Petunia occidentalis had the longest pollen tubes after 6 h of culture but had a relatively shorter style. Conversely, the pollination experiments revealed that P. axillaris subsp. parodii, which had the longest style, produced the longest pollen tubes in vivo. The results revealed no clear relationship between the style lengths and the growth rate of pollen tubes in vitro. Interspecific pollinations indicated that the styles affected pollen tube growth. We concluded that, in vitro, the pollen tubes grow without being affected by the styles, whereas, in vivo, the styles significantly affected pollen tube growth. Furthermore, interspecific pollination experiments implied that the pollen tube growth tended to be suppressed in the styles of self-incompatibility species. Finally, we discussed the pollen tube growth ability in relation to style lengths.     

欧报春二型花柱的繁育特性研究

二型花柱植物通常具有自交不亲和性,在不同植物中所表现的可育性不同。欧报春(Primula vulgaris)是典型的二型花柱植物,为了探究欧报春的繁育特性,通过温室栽培,对欧报春的长花柱和短花柱的花部特征、花粉和花柱形态、花粉活力、柱头可授性、花粉/胚珠比、杂交指数、杂交亲和性、花粉管观察进行研究。结果表明,(1)长花柱和短花柱的花冠直径、花冠筒长度、柱头高度、花药高度和花筒中部直径均表现出两型性;(2)花粉和柱头观察发现花粉极轴长、花粉赤道轴长、花柱直径、柱头乳突细胞和花粉数量均有差异;(3)长花柱的花粉/胚珠比为384.20,短花柱为369.70,属于兼性异交类型;长花柱和短花柱的花粉活力和柱头可授性能在较长时间内维持较高活力;(4)长花柱杂交指数值为5,短花柱为4,表明繁育系统类型为异交,部分自交亲和,需要传粉者;6种授粉组合均能结实,异型花间授粉的结实数明显高于同型自花授粉和异株同型授粉;短花柱为母本的异型花间授粉组合亲和性优于其他组合;欧报春存在自交不亲和性,长花柱的自交不亲和性低于短花柱。     

Adhesion of lily pollen tubes on an artificial matrix

 We proposed that pollination in lily is a case of cell adhesion and cell movement, but experimental evidence for the adhesion event is lacking. In this study, we developed an artificial extracellular matrix that mimics the in vivo lily stylar transmitting tract. This artificial matrix was created by applying the transmitting tract exudate extracted from lily styles onto a nitrocellulose membrane. When in vitro-grown pollen tubes were applied to the matrix, they adhered by their tips to the area of the stylar exudate which is rich in arabinogalactan proteins. Once they adhered, they grew on the in vitro artificial matrix at rates faster than normal. This is the first experimental evidence demonstrating the adhesion of in vitro-grown pollen tubes, an event that has been described as common in vivo. The adhesion event is stylar exudate specific, concentration dependent, and is affected by the developmental age of the pollen tube. This bioassay for pollen tube adhesion will be used to isolate the adhesive molecules from the stylar exudate. Received: 9 December 1996 / Revision accepted: 5 May 1997     

Polyester and nylon powders used as pollen diluents preserve pollen germination and tube growth in controlled pollinations

Pollen acquisition for seed production, breeding programs and supplemental pollination can be costly and difficult. The identification of dry particulates for use as pollen diluents would facilitate the use of limited amounts of pollen and aid in accurate pollen application and dispersion. Four powders - Rilsan ES, polyester, wheat flour, and Lycopodium spores - were evaluated as pollen diluents using petunia as a model system. Diluents were combined with petunia pollen at a 5:1 (v/v) ratio. Two types of studies were conducted: (1) storage studies evaluated the viability of pollen combined and held with diluent for different durations; and (2) in vivo studies evaluated pollen tube growth in the styles of flowers pollinated with pollen-diluent mixtures. Pollen germination was not affected when stored as pollen-diluent mixtures for 4 days. Slight detrimental effects on pollen germination were observed after 6 days storage with Rilsan ES powders. Pollination with all the pollen-diluent mixtures resulted in fewer pollen tubes growing in the style compared to controls diluted with heat-killed pollen instead of diluent powders. Lycopodium-pollen mixtures were the most inhibitory, providing only 8% of the tube numbers observed in controls. Pollen mixed with polyester powders, Rilsan ES powders or wheat flour had tube numbers ranging from 47 to 61% of the control, but still had 175 or more pollen tubes per style, which would be sufficient for high rates of seed set in petunia. Wheat flour-pollen mixtures tended to clump and degrade pollen flow. Rilsan ES and polyester were identified as two promising pollen diluent powders that can facilitate accurate metering and distribution of pollen, produce large numbers of pollen tubes, and maintain pollen viability under storage.     

Pollen tube growth in pistils of female-sterile and fertile plants ofOenothera mut.brevistylis

Summary Pollination and pollen tube growth was observed in pistils of fertile and female-sterile plants ofOenothera mut.brevistylis. The stigmas and styles of sterile plants were very irregularly formed and additional ovules often developed in the style. The malformations did not inhibit pollen tube growth but made it slower and less efficient. The pollen tubes grew in the ovary but did not find the ovules. Near their tips, pollen tubes branched or meandered without any clear direction. Lack of tropism caused by malfunction of the ovules is postulated in the female-sterile plant.     

Pollination-Induced Ethylene in Carnation (Role of Pollen Tube Growth and Sexual Compatibility)

The pollen-pistil interactions that result in the stimulation of ethylene production and petal senescence in carnation (Dianthus caryophyllus L.) flowers were investigated. Pollination of White Sim flowers with Starlight pollen elicited an increase in ethylene production by styles, leading to increased petal ethylene and premature petal senescence. In contrast, pollination with 87-29G pollen led to an early increase in ethylene production, but this was not sustained, and did not lead to petal senescence. Both Starlight and 87-29G pollen germinated on White Sim stigmas and their tubes grew at similar rates, penetrating the length of the style. Crosses between Starlight and White Sim led to the production of viable seeds, whereas 87-29G pollen was infertile on White Sim flowers. Pollination of other carnations with 87-29G elicited ethylene production and petal senescence and led to the production of viable seeds. These results suggest that physical growth of pollen tubes is insufficient to elicit a sustained increase in ethylene production or to lead to the production of signals necessary for elicitation of petal ethylene production and senescence. Rather, the cell-cell recognition reactions leading to sexual compatibility in Dianthus appear to play a role in this interorgan signaling after pollination.     

Covalently bound wall proteins of pollen grains and pollen tubes grown in vitro and in styles after self- and cross-pollination in Lilium longiflorum

Summary A method was worked out using trifluoromethanesulfonic acid (TFMS) as a reagent to split the covalently bound proteins, which are NaCl insoluble, from pollen tube walls of Lilium longiflorum, leaving the peptide bonds essentially intact. After electrophoretic separation, comparisons were made among these proteins from pollen grains and pollen tubes grown in vitro and in styles after self- and cross-pollination. It was found that a) the patterns of covalently bound wall proteins were different between tubes grown in vitro and in vivo; b) fewer bands were found in covalently bound wall proteins than that in noncovalently bound proteins; c) the bands remained almost the same no matter whether the tubes had been cross pollinated or self pollinated, indicating that while the noncovalently bound proteins were involved in incompatibility as shown in the previous paper, the covalently bound proteins may only serve as a structural component, having little to do with incompatibility.     

A pollen tube growth-promoting arabinogalactan protein from nicotiana alata is similar to the tobacco TTS protein

Upon germination on the stigma, pollen tubes elongate in the stylar transmitting tract, aided by female factors, with speed and directionality not mimicked in in vitro pollen tube growth cultures. We have shown that a stylar transmitting tissue arabinogalactan protein (AGP) from Nicotiana tabacum (tobacco), TTS protein, stimulates pollen tube growth in vivo and in vitro and attracts pollen tubes grown in a semi-in vivo culture system. It has been reported that the self-incompatible Nicotiana alata produced a stylar glycoprotein, GaRSGP, which had a backbone polypeptide that shared 97% identity with those of TTS proteins but some of its properties were different from those described for TTS proteins. We report here the characterization of a family of stylar transmitting tissue glycoproteins from N. alata that is virtually identical to tobacco TTS proteins and which we refer to as NaTTS proteins. Like their tobacco counterparts, NaTTS proteins are recognized by the traditional AGP-diagnostic reagent beta-glucosyl Yariv reagent, and they are also recognized by JIM13, a monoclonal antibody against AGP. NaTTS proteins also stimulate pollen tube elongation in vitro and attract pollen tubes in a semi-in vivo pollen tube culture system. Biochemical and immunological characterization of NaTTS proteins revealed that they have extraordinary variability in the extent of sugar modifications of their polypeptide backbones. The extent of sugar modifications on NaTTS proteins significantly affects their biochemical properties, influences how they interact with the transmitting tissue extracellular matrix, and affects their solubility from this matrix. Our results suggest that the strategy used to purify GaRSGP only recovered a less glycosylated, more tightly extracellular matrix-bound sub-population of the entire spectrum of N. alata TTS proteins.     

烟草传粉前后生殖器官中细胞分裂素含量的变化

经免疫亲和层析系统纯化后,用间接竞争ＥＬＩＳＡ 法测定了烟草（Ｎｉｃｏｔｉａｎａ ｔａｂａｃｕｍ ）生殖器官在传粉前后细胞分裂素（ｔ－ＺＲ,ｉＰＡ）含量的变化． 开花前５ ｄ,花药和花丝中的细胞分裂素（ＣＴＫ）含量均达到最高值,以后随雄蕊发育逐渐下降． 授粉使花柱ＣＴＫ 含量急剧上升,授粉后１ ｄ 达到高峰,未经受粉的花柱ＣＴＫ 开花后下降．授粉后２ ｄ,子房中的ＣＴＫ 开始上升,在授粉后４ ｄ 达到最高值,而未受精的子房ＣＴＫ 含量开花后下降． 传粉后雌蕊中ＣＴＫ含量随花粉管生长而有规律地增加,ＣＴＫ 积极参与植物的传粉和受精过程     

Change in Cytokinin Levels in Reproductive Organs of Nicotiana tabacum Before and After Pollination

Immunoaffinity systems and indirect competitive ELISA were used to determine the changes of cytokinin (CTK) content in reproductive organs of Nicotiana tabacum before and after pollination. It was found that t-ZR and iPA existed in all reproductive organs tested, and their contents were higher in female than in male organs. On the 5th day before anthesis. CTK reached its peak value in anthers and filaments, then it declined. In styles, CTK content increased after pollination and reached its peak one day later. In ovaries, levels of CTK began to increase two days after pollination and reached its peak two days later, just at the time when fertilization took place. If unpollinated, CTK levels decreased both in styles and ovaries. Pollination and fertilization resulted in an increment of CTK in pistils, accompanied with the process of pollen germinfition and pollen tube growth. It could thus be inferred that t-ZR and iPA played some active role in pollination and fertilization.     

不同葡萄品种柱头、花柱发育与种子形成的关系

对‘京秀’、‘香妃’(二倍体)和‘黑奥林’、‘巨峰’(四倍体)葡萄品种的柱头、花柱发生发育以及花粉管在雌蕊中生长与坐果和果实中种子形成的关系进行了研究。结果显示:‘京秀’、‘香妃’的坐果率和果实中的种子数明显高于‘黑奥林’和‘巨峰’,而‘巨峰’葡萄的坐果率和种子数最低。开花期‘京秀’和‘香妃’的花柱直径略小于‘黑奥林’和‘巨峰’,但花柱中部引导组织的直径和引导组织所占花柱的比例却大于‘黑奥林’和‘巨峰’;半实心花柱的比例也高于‘黑奥林’和‘巨峰’,说明花柱中引导组织的结构和发育状况可能与坐果和果实中种子形成有关;人工授粉24 h后,在‘京秀’和‘香妃’雌蕊中花粉管的生长速度快于‘黑奥林’和‘巨峰’,授粉后48 h‘京秀’和‘香妃’雌蕊中到达各部位的花粉管数要多于‘黑奥林’和‘巨峰’,授粉后72 h各品种到达子房各部位的花粉管数开始减少。表明‘京秀’和‘香妃’花柱中引导组织较为发达,而且半实心花柱比例也较高,有利于花粉管在雌蕊中的生长和完成授粉受精,使‘京秀’和‘香妃’的坐果率和果实中种子的形成都较高。