In this article we will discuss about the events in pollen-pistil interaction with its significance.
Pollen-Pistil Interaction:
Pollen grains are deposited on the stigma either due to closeness of the anthers to the stigma or by pollinating agents (biotic or abiotic). This unique feature brings about pollen-pistil interaction between the male gametophyte, the pollen grains, with the massive sporophytic tissue.
A successful pollination brings about sequential events in the pollen-pistil interaction that ultimately ends up by the discharge of the male gametes in the embryo sac (Fig 6.1).
All the events beginning from pollination to the release of gametes in the embryo sac form a part of the pollen-pistil interaction or the programic phase.
Pollen Attachment and Hydration:
The attachment of the pollen on the stigma depends upon its wall sculpture and stickiness. In wet stigma adhesion is mostly a mechanical process, whereas, in dry stigma it depends on the extent and composition of the pellicle, and the amount of surface-coat substances on the pollen.
Pollen hydration proceeds in a controlled manner characterized by distinct area of stability of increasing water content and can begin in the anther before pollen release. Its rapidity is dependent to a great extent on the nature of stigma, for instance in a dry stigma hydration is gradual and controlled by the water potential of the stigma and pollen.
This controlled hydration provides suitable conditions for the recovery of the membrane integrity of the vegetative cell.
A plausible pathway for hydration in dry stigma as proposed by J.Heslop-Harrison (1979) is given below:
In a stigma with aqueous exudates hydration is very rapid. For instance in Petunia the stigma is covered with a lipoidal exudates and a thin layer of water which establishes a moisture gradient through the lipoidal exudates. The pollen grain thus gradually gets hydrated.
Ultrastructural and physiological studies of pollen hydration in Brassica show two distinct phases of hydration. During the initial phase, putative signals are reciprocally exchanged between pollen and stigma. The second phase proceeds with an invagination of the intine in the colpial zone and formation of a ‘foot’ of pollen coating that contact the stigma papilla.
Freeze-etch preparation show microchannels at the papilla-pollen boundary through which water moves from stigma to pollen grain but not between grains. The area around the site of pollen tube emergence is rich in pectins, and one of the earliest visible alterations of macromolecules upon hydration is a loss of protein and pectic material from the length of the colpial slit.
Pollen Germination and Tube Growth:
The stigmatic surface provides the essential prerequisites for a successful germination that are absent in the pollen. In wet stigma, the role of the stigmatic exudates in pollen germination is highly variable.
In Amaryllis and Crinum, stigmatic exudates are essential for pollen germination, however, in Nicotiana and Petunia the exudates play no significant role during germination, since young stigmas free from exudates support satisfactory germination of pollen grains.
In dry stigma, the pellicle plays a vital role in germination. Its enzyma tic removal inhibits pollen germination or pollen tube entry into stigma. For instance in Raphanus sativus enzymatic digestion of the pellicle reduces pollen germination and totally inhibits the entry of even compatible tubes into the stigmatic papillae.
The stigmatic surface also provides boron and calcium which are required for germination but are deficient in pollen. It has been seen that those stigmatic secretion of Vitis vinifera that contain 2-5 ppm of boron permit pollen germination.
The growth of the pollen tube of flowering plants is restricted exclusively at their apices. Microscopic examination of growing pollen tubes reveals that most of the cytoplasm is restricted to the apical region while a large vacuole fills the grain and the older region of the tube (Figure 6.6). The cytoplasm is restricted to the apical region of the growing tube by the formation of series of callose plugs at regular intervals behind the tip (Figure 6.7).
The callose plugs are formed as a ring on the inner side of the tube wall and gradually grow toward the centre which finally seals off the growing tip from rest of the pollen tube. There is characteristic zonation, in which the apical region of the tube possesses a clear cap called “cap block” with more granular elements behind. The “cap block” disappears with the termination of the growth. These internal components exhibit vigorous “reverse fountain” cytoplasmic streaming.
However, within the tip itself the motion is chaotic and turbulent, with vesicles appearing to move in a random, from the base of the clear zone to the extreme apex. There is marked accumulation of secretory vesicles often in the shape of an inverted cone at the tube apex.
These vesicles contain components for cell wall expansion, because more vesicles are secreted than are required to support the increased area of the plasma membrane. Actin polymerization is necessary for pollen tube growth.
Actin microfilaments (MFs) are involved in the transport of secretory vesicles essential for cell elongation and is accumulated in substantial amounts in mature pollen grains. The cytoplasm behind the tip is rich in cell organelles, lipid bodies, vesicles, and amyloplasts.
Pollen tubes do not grow uniformly, but rather in bursts or pulses. In Petunia and tobacco, the tube cell elongates with alternating bursts of fast and slower growth, while lily tubes, especially those longer than 700 pm, grow with a periodic oscillatory pattern in which the rate changes in smooth sine wave. In lily pollen tubes the rate changes from 100-500 nm/sec with a frequency of 15-50 sec.
Many underlying physiological processes also oscillate with the same frequency, but with varying phase relationships to growth rate. For example, the intracellular Ca2+ gradient oscillates in phase or slightly behind the growth peak, while the extracellular Ca2+ influx exhibits a 10-15 sec delay. H+ also oscillates.
To date, genes specifically associated with pollen germination have not been identified. However, the large number of unidentified proteins (> 230) whose appearance is coincident with germination suggests that it may be premature to conclude that none of them arises from transcripts activated specifically at germination.
Some early gene products such as alcohol dehydrogenase, actin, and a heat- shock protein from tomato persist in germinating pollen. Although the late genes are transcriptionally activated before dehydration, their persistence during germination and growth argues for a functional role at this stage.
A pollen specific calcium-dependent calmodulin-independent protein kinase (CDPK) isolated from maize suggests the presence of post-translational control mechanism involving Ca2+ and phosphorylation. The gene is transcribed in mature and germinating pollen and is required for germination.
Following pollen germination the pollen tube-grows on the surface of the stigmatic papillae, e.g., Gossypium, or through the cellulose-pectic layer of their walls, e.g., Lilium. The stigma provides the pollen with water and necessary medium in the form of exudates for its germination. The exudates are highly viscous, refractive and adhesive. These are rich in lipids, small amount of free sugars, amino acids, proteins, and peptides.
In dry stigma and solid style, the pollen tube degrades the cuticle by cutanase released by the pollen. Pollen grains contain an elaborate set of enzymes and some of these are available as soon as the pollen grain makes contact with the stigma.
The digestion of the cuticle allows the tube to enter the pectocellulosic wall of the papillae and finally grow through the intercellular substances of the stigma and the style. In Gladiolus the pollen tube grows through a mucilaginous substance accumulated between the cuticle and cell wall, instead of the pectocellulosic wall.
In wet stigma and solid style the cuticle gets disrupted during the secretion of the exudates and the pollen tube enters the intercellular matrix of the stigmatic tissue.
Path of Pollen Tube in the Style:
In species with wet stigma and solid style the cuticle of the stigma /papillae is disrupted during the secretion of the exudates, thus there is no physical barrier for pollen tube entry into the intercellular spaces of the transmitting tissue of the stigma. In taxa with wet stigma and hollow style, pollen tubes grow on the surface of the stigma and enter the stylar canal.
In species with dry stigma and solid style the cuticle provides the physical barrier for the pollen tube entry. The cuticle is eroded at the point of contact by the activity of cutinases released by the pollen. After the digestion of the cuticle, the tube enters the pectocellulosic wall of the papillae and finally grows through the intercellular substances of the stigma and the style.
In Gladiolus and Crocus the pollen tube grows through the mucilage accumulated between the cuticle and the cell wall.
Pollen tube growth is a calculated directional cell migration, along the transmitting tissue of the style. In most of the species pollen tube make their way to the ovary through the intercellular matrix of the transmitting tissue or through the mucilaginous matrix of the hollow style. The secretion product of the glandular cells of the solid stylar tissue is deposited in the matrix.
It is a heterogenous mixture consisting chiefly of sugars, proteins, gycoproteins, and lipids. In several dicotyledons and monocotyledons the transmitting tissue contains arabinogalactan proteins. This glycoprotein is style-specific, and its presence in the cytoplasm and cell walls of compatible pollen tubes growing in the style have favoured its role in the nutrition, growth, and guidance of pollen tubes in the stylar tract.
In the hollow styles, the canal cells secrete a mucilaginous substance that later forms an extracellular matrix and accumulates in the stylar canal. The major component of this secretion is again an arabinogalactan protein.
Path of Pollen Tube in the Ovule and Embryo Sac:
The pollen tube finally pushes through the ovule and reaches the embryo sac. This guidance into the ovule is in terms of essential signals originating from the male and female tissues.
Evidences obtained from the analysis of developmental mutants of Arabidopsis, viz., bell and sinl (where integument and embryo sac development in the ovule is affected) suggests that genes active in the female gametophyte play a crucial role in the signalling process that guides pollen tubes to the ovule.
In fact pollen tubes are always attracted to ovules with a functional embryo sac, which confirms a female gametophytic control of pollen tube guidance.
Recognition of the Pollen by the Stigma:
The stigmatic surface of a flower provides refuge to various pollen grains, but a physiological mechanism operates to ensure that only intraspecific pollen germinate successfully. In sporophytic self-incompatibility system the recognition reaction system sets in almost immediately after the pollen comes in contact with the stigma.
This is also true for certain gametophytic self- incompatibility systems. The components of pollen recognition system are present in all floral organs, but are segregated to the surface of the stigma by the action of genes like PDH.
Recognition of compatible pollen grains by the stigmatic papillae involves a molecular interaction between substances present in the pollen wall and those present on the pellicle or in the stigmatic surface.
In fact recognition mechanism is switched on with the hydration of pollen and the subsequent release of its wall proteins. The molecular events that are switched on, following the acceptance or rejection of the pollen grains have been studied in Brassica napus, B. oleracea, and Arabidopsis, from where the details are gradually emerging out.
The pollen grains following contact with the stigma synthesis nearly forty new proteins, and few of these proteins are highly phosphorylated. Thus it appears that protein phosphorylation may be responsible for signal transduction in a compatible association.
Moreover, there is a brief Ca2+ peaks in the stigmatic papillae of Brassica napus following its contact with compatible pollen grains. Thus there is a definite participation of a Ca2+ in pollen signal perception but how the system is activated to promote hydration and germination of pollen grains is not clear.
The involvement of a specific component of the pollen-wall-based tryphine in the regulation of pollen-stigma interactions has been provided by the mutational study in Arabidopsis. Preuss (1993) isolated a mutant (pop1) Arabidopsis, which was defective in pollen-pistil interactions, thus inducing male sterility.
The grains failed to hydrate on the stigma and germinate, though it responded successfully in vitro. The failure of the mutant to germinate in vivo was possibly due to loss of tryphine components on the wall of mutant pollen grains, and this was substantiated by the absence of wax on the stem of pop1 mutants.
Further chemical analysis has shown that wax deficiency on the stem is correlated with the absence of long chain lipids on the mutant pollen grains. Further a mutation in the CER1 locus of Arabidopsis which affect pollen hydration on the stigma, have also been traced to lack functional tryphine layer with the normal components of lipids on the pollen grains.
The gene has also been shown to encode for a new protein, involved in catalytic steps of wax biosynthesis. Thus lipid molecules and tryphine are involved in the signaling mechanism that allows the stigma to have a successful pollination event.
Pollen grains of a transgenic Petunia hydrida plant, deficient in flavonoids failed to germinate or produced very short pollen tube in vitro. This inhibition was nullified by the addition of stigma extract from the wild type Petunia or by adding micromolar quantities of flavonoids (kaempferol) to the germinating medium.
Sharma and Shivanna (1983 a, b) investigated the biochemical basis of self- incompatibility recognition in Petunia hybrida by using in vitro assay. Pollen grains were cultured in a medium containing pistil extract and before that the grains were treated with sugars or with lectins.
Interestingly self-incompatibility could be avoided when the pollen grains were treated with glucose or lactose but not with lectins thus indicating that lectin-like components are involved in self-incompatibility recognition. Blocking these molecules with corresponding sugars make them ineffective in establishing recognition, and thus overcome inhibition.
Likewise, inclusion of specific lectins into medium containing pistil extract was also effective in overcoming inhibition of self-pollen. Seemingly, recognition factors in the pistil are polysaccharide-containing molecules and the binding of the saccharides with the specific lectins makes them ineffective in recognizing self-pollen.
Significance of Pollen Pistil Interaction:
Pollen-pistil interaction is unique in flowering plants and plays a significant role in sexual reproduction and seed formation. The pollen grains carrying the male gametes do not have a direct admittance to the female gamete.
Thus they need to be deposited on the stigma and it’s pollen tube has to grow through the massive sporophytic tissues of the stigma and style prior to release of male gametes near the egg. The pistil has developed a unique mechanism to recognize pollen grains and thereby permit the growth of pollen tube in compatible cases.
Incompatible tube is thus effectively prevented from reaching the embryo sac. The barrier to fertilization is restricted to the sporophytic tissues of the pistil. At the same time there is no chance of unsuccessful fertilization ones the male gametes have been released near the egg.
The principal significance of pollen-pistil interactions are:
a. The most essential requirement for sexual reproduction is the screening and selection of male gamete and this is achieved during pollen pistil interaction. Thus, pollen-pistil interaction offers enormous potential for the manipulation of pollen screening which is obviously for the quality and compatibility of pollen.
b. The number of pollen grains that are generally deposited on the stigma under normal conditions are far greater than the number of ovules available for fertilization. As a result the pollen grains are subjected to a tough competition during pollen-pistil interaction.
Only those pollen that germinate early and have a faster growing pollen tube, i.e., more vigorous, are able to withstand the rigidity of post- pollination competition and fertilization. Consequently competition among pollen grains during pollen-pistil interaction results in the increased vigour of the progeny. Thus this interaction can be considered as an important contributory factor in the evolutionary success of flowering plants.
c. It has a direct relevance to plant breeding programmes. A plant breeder continuously strives to bring together desirable characters present in different taxa, through hybridization.
Thus a better understanding of the biology of pollen-pistil interaction would no doubt, enable the plant breeders to manipulate the screening process in the pistil more effectively.