2.1. Proteome Dynamics in Pollen at Different Points from Development to Germination
Before cell-to-cell interaction, pollen has to undergo a complex and necessary development to form a suited grain with high quality. Anther with four locules is the progenitor cells to release microspores, which is generated from microsporocyte via two continuous meiotic divisions [
12]. The microspores released in the locule undergo cytoplasmic reorganization mediated by the cytoskeleton, and it also termed as polarizability. Subsequently, the polarized cell forms a male germ unit not only with two sperm cells that fuse with egg cell and central cell, respectively, but also with a vegetative nucleus via two mitotic divisions. The pollen with the three cells is called mature pollen, which can interact with gynoecia and be selected into the style for tube growth to access the embryo sac for double fertilization [
13,
14]. Mature pollen germinates fast after pollination from quiescent to active state, and the vegetative cell grows a pollen tube transporting the sperm cells into the embryo sac to complete the double fertilization process.
According to previous study on the pollen transcriptome in the model species
Arabidopsis, 992 pollen-expressed mRNAs were identified by comparing with that of the sporophyte. Among them, nearly 40% were detected specifically in mature pollen, encoding proteins involved in carbohydrate metabolism and cytoskeleton dynamics, which would be a key to ensure the quality of pollen through regulatory and functional specialization [
15]. Furthermore, one-third of the genes constitutively expressed in the vegetative tissues were not expressed in pollen. These results revealed that the transcriptome of gamete showed more simplicity and higher proportion of selectively expressed genes than sporophytic tissues [
16]. Soon afterwards, a proteome map for mature pollen of
Arabidopsis thaliana was drawn using 2-DE followed by ESI-MS/MS to expound the biochemistry of pollen [
17]. Among the 135 identified proteins, there were almost 20% involved in metabolism, 17% in energy generation, or 12% in cell structure, which is similar to the study at transcriptional level. Moreover, seven proteins whose RNAs were not shown in the transcriptome have functions in metabolism, energy generation or cell structure. To resolve the omission of short and low abundant proteins, a new generic deterministic peptide classification scheme was set up to identify the proteins with minimized error rate on the Arabidopsis pollen [
18]. In addition to the large scale on the pollen grains, the analysis on the pollen coat at gene level and protein levels had been taken [
19]. There were 322 special proteins in mature pollen of rice using MS technologies, which had been classified into at least 14 functional categories [
20]. Among them, 38 unique proteins were beta-1,4-xylanase and beta-glucanase in pollen coats, which were major proteins in the pollen coat of maize [
21]. The released proteins from the pollen might contribute to the germination and growth of the grains and pollen tube.
In a way, the revelation of the gene level cannot directly link to the discipline of the protein level and activity. The proteomics work on developmental pollen used two-dimensional gel to find disparate points and then identified them by MALDI-TOF MS based on the sequence dates. For example, in
Oryza sativa, the comparative proteins of the different stages of pollen (pollen mother cell, tetrad, early young microspore, middle young microspore, early binucleate, late binucleate, heading stage) were taken into analysis by similar proteomics technologies. The 33 unique proteins with the same changing trend participated in sugar metabolism, cell elongation and expansion, which were essential to the pollen germination [
22]. Analogously, rice mature (MPG) and germinated (GPG) pollen grains were selected artificially and then flowed to 2D-gels to obtain protein spots. Comparing proteins of the two different growth stages, 186 proteins from almost 2300 proteins were differentially expressed. These proteins are involved in regulatory and metabolic processes, such as the dynamics of protein and cytoskeleton [
23]. Comparing the protein expression of the germinating pollen (GP) and the mature pollen (MP) of canola (
Brassica napus) via DIGE associated to MALDI-TOF/TOF, the up-regulated proteins play roles in carbohydrate, nucleotide and protein metabolism, signal transduction and stress responses. On the contrary, some catalases and LEA proteins were showed to be down-regulated. These showed that the proteins associated in macromolecules’ metabolism and enzymes involved in the signal pathways were essential to the pollen germination [
24]. The differentially expressed proteins in pollen tubes compared to the un-germinated pollens could demonstrate the special physiological processes that occur during the development of the pollen tubes [
25]. Recently, a proteomics analysis was also conducted in pollen (before-pollinated and/or pollinated) and pollen tube of the
Picea meyer [
26],
Triticosecale wittmack [
27] and
Lilium davidii [
28]. These researches are better conducted in two aspects: pollen collection, including the condition of pollen grains germination
in vitro, the nutrition and temperature for plants growth, pollen review by light microscopy, and the identification and quantification of proteomics, covering the ITRAQ labeling method and MALDI analysis by TOF/TOF instrument. The proteomics for male gametophyte contribute a new slight on examining the molecular mechanism and lay a great foundation for the study of fertilization.
2.2. Protein Analysis on Pistils by Comparative Proteomics
Pistil, another participant, is composed of stigma, style, and ovary [
29]. The apical stigma is in the position of capturing and ingesting pollen grains. The style can be regarded as a subtle bridge linking the stigma and the ovary, where the transmitting tissue plays role accompanying the extension of the pollen tube. The basal ovary is an organ containing the ovules, which gestate the embryo sac to grow the eggs [
30,
31]. As the name suggests, pollination depends on transferring male gametophyte (pollen grain) from the anther to the sigma of pistil. That is the space where interaction occurs. Judging by whether or not there is secretion on the surface, the stigmas can be classified into two broad categories, wet and dry. Comparing the two biochemical reactions based on the different structure, up to date, pollen capture by the wet stigmatic secretion is nonspecific while the recognition process shows a degree of species specificity in the dry-type stigma. Besides, pollen hydration within the secretion is passive and unregulated. On the contrary, it is a regulated process in dry stigma [
2].
Crucifer is the typical family with dry stigma, which is selected as model plants for study of pollen-pistil interaction. Through comparing the whole-genome transcriptional profiles of stigmas and ovaries isolated separately from wild-type
Arabidopsis and transgenic plants, in which cells of the stigma epidermis and transmitting tract were ablated by expression of a cellular toxin on the microarray platform, 115 and 34 genes were identified from 23,000 genes on the array to be expressed specifically in the stigma epidermis and transmitting tract. The proteins encoded by these genes were functional classified in signal transduction pathway, regulating the components of the extracellular matrix during pollination. Among them, S-locus receptor kinase (SRK), M-locus protein kinase (MLPK) and arm repeat containing (ARC1) play roles in self-incompatibility [
32].
The affymetrix ATH1 whole genome array were used in comparing the different gene levels in un-pollinated pistils and un-fertilized ovules of
Arabidopsis thaliana, as well as the pollinated pistils in special timing points that represented the most significant development from pollination to fertilization. The result showed 1373 genes were differentially expressed during pollen–pistil interaction, whose function were explained and projected to the extent necessary for successful fertilization [
33]. The temporal and spatial gene expression profile of
in vivo pollen-pistil interaction provided a detailed evidence of changing in gene expression pattern, further supporting the molecular mechanisms operating during pollination.
Recent researches on proteomic analysis of the pistil in crucifer were mainly taken in SI response to find the candidate proteins. This implies that the mechanism of compatible interaction between pollen and pistil is not still directly addressed, especially the proteome in pistil. After searching—a comparative proteomic analysis of pistil before and after pollination in Soybean cooperated with the proteome database [
34] and transcriptomics analyses [
35]—a strict self-pollination plant was found. According to the MALDI-TOF-MS results based on the 2D-gel, 58 differently expressed proteins were identified, of which there were 22 up-regulated proteins and 36 down-regulated proteins after pollination. After functional classification, the largest group was metabolism-related proteins. Among them, the sucrose-phosphate synthase (U18) was increased in expression, while some isoforms of glutamine synthetase were decreased. These indicated that the primary metabolisms were enhanced to facilitate the pollination and the following pollen tube growth [
36]. This study enhances our understanding of the level of proteins expression, as well as the participated biological processes.
The representative plants with the wet stigma are mainly Solanaceae, Rosaceae and Liliaceae [
2]. The stigmatic secretion (SE) on the surface of the stigma plays an important role in ingesting the compatible pollen grains. Except the lipid and carbohydrate, SE also contains a wide range of proteins with profound functions, such as the sigma-specific protein 1 (STIG1), regulating the timing of the accumulation of SE in tobacco and petunia [
37]. The first analysis of SE proteins on a large-scale level was taken in the
Lilium longiflorum and
Olea europaea by SDS-PAGE coordinated with the LC-MS/MS. However, given the un-completely genome annotation of the two plants, a database search algorithm (Mascot) was employed and identified 51 proteins in Lily and 57 in olive, of which only 13 were present in both SEs. In-depth analysis of these proteins showed that more than half of the proteins contain a signal peptide, and it was predicted that the SE might participate in at least 80 different biological processes and 97 molecular functions, of which included the carbohydrate metabolism, cell signaling and response to the biotic and abiotic stresses. During pollination, the catabolic enzymes disintegrate large polysaccharides and lipids into smaller units to regulate pollen tube growth by selective degradation of cell-wall polysaccharides. Two Stigma/stylar cysteine-rich adhesion (SCA) isoforms, a chemotropic protein and a fasciclin-like domain (FAS) protein were identified in the lily SE with the role of pollen tube adhesion [
38]. By proteomics, a comprehensive map of proteins can be built to discover their biological function within pollination.
When the male and female gametes are prepared for the next journey, the recognition and interaction will induce the tube growth via compatible signal and chemical gradients. The extension keeps until the tube enters into the embryo sac, where the molecules and receptors of tube response with the guidance from the female gametophyte [
39]. Comparing to angiosperm, the pollination droplet is a unique and conservative pollination mechanism in gymnosperm. Proteomics analysis and identification taken on the mechanism revealed the proteins participating in the drop were related to the pathogen defense and pollen development [
40]. Although there have not been a complete network for the interaction between the tube and pistil, some key regulators and receptors have been identified by multidisciplinary approaches including biochemistry, molecular genetics and functional genomics. On the contrary, in
Arabidopsis, the female gametophytic guidance is divided into two stages: the funicular guidance, implying that the tubes extend from the septum to the funiculus, and the micropylar guidance, implying that the tube navigates from the funiculus to the female gametophyte through the micropyle [
41]. For example, MYB98, as a transcription factor, expresses in the synergid cell as micropyle guidance [
42,
43]. AtLURE1 peptides belong to the defensin-like (DEFL) peptides, expressed in synergid cells and secreted toward the funicular surface to guide the tube growth as especially guidance [
44]. Looking back on how the receptors on the pollen tube interact with the female guidance, the ion gradient regulated by the transmembrane proteins and channels are critical for the tube growth, such as the cyclic nucleotide-gated channel 18 (CNGC18), working as a channel to regulate the Ca
2+ concentration, and a GABA transaminase, which is coded by the
pollen pistil 2 (
POP2) and has the function to increase content of GABA in the style and ovary [
39,
45]. Besides, lost in pollen tube guidance 1 (LIP1) and 2 (LIP2) were verified as a receptor complex to respond to AtLURE1 [
46]. Especially, the mitogen-activated protein kinase 3 (MPK3) and the mitogen-activated protein kinase 6 (MPK6) were verified with the function of guiding the direction to the right towards the mutational plants of mpk3 and mpk4 [
47].
Although much research had been conducted on several model species around compatible and even incompatible pollination and fertilization, there was a common theory among the types of molecular regulation on the pollen-pistil interaction [
48]. Researchers suggested the absence was as a result of rapid evolution and change of proteins regulating sexual reproductive process [
49]. For instance, arabinogalactan proteins (AGPs), a family of hydroxyproline-rich glycoproteins (HRGPs), showed an increased expression during pollination in the olive pistil (
Olea europaea L.) compared to the non-pollinated pistil, while the expression of AGPs decreased after pollination. AGPs were localized predominantly in the cell wall of secretory cell of the stigma, as well as in the transmitting tissue of the pistil during the pollination period by means of immunofluorescence localization. These results proved that proteins play roles of supporting pollen performance and tube growth during the pollination stage in olive, which corresponds to the conclusion that AGPs had roles in vegetative, reproductive, and cellular growth and development, previously [
50,
51].