Sporopollenin: Distribution, Production and Functions

In this article we will discuss about:- 1. Distribution of Sporopollenin 2. Chemistry of Sporopollenin 3. Production and Deposition 4. Functions.

Distribution of Sporopollenin:

It was first observed and named as “sporonin” by John (1814) and latter characterized by Berzelius (1830). Fossil green algae dating back to Devonian period have been shown to contain sporopollenin. The oldest sporopolleninous acritarchs occur in Pre-Cambrian rocks, 1.2-1.4 billion years old.

The green algae are presumably responsible for the development of sporopollenin and its introduction into the armament of higher green plants, where its principal function is protection against oxidation and desiccation.

Further Brooks and Shaw (1968,1971) from their study of the Pre-Cambrian rocks of Onverwacht, 3.7 b.y. old, and the Fig Tree cherts of South Africa, 3.2 b.y. old, have shown the presence of amorphous insoluble organic material which appears similar to present day sporopollenin.

It forms the basic structure of the resistant wall of most palynomorphs, like spores, pollen, dinoflagellates, and acritarchs. It has also been recorded from the spores of Aspergillus niger, sexual (±) spores of Mucor mucedo, asexual spores of Pithophora oedogonia and several algae like, in the cell wall of Phycopeltis epiphyton (a subaerial green alga found growing on the leaves of vascular plants and bryophytes), Char a corallina, cyst of Prasinocladus marinus.

A trilaminar sporopollenin sheath is also present in Chlorella, Scenedesmus, and Pediastrum. It is also distributed in the spores of Bryophytes, Pteridophytes, pollen of Gymnosperms and Angiosperms.

In general it is limited to the outer wall, the exine however, fern spores and some gymnosperm pollen have an additional sporopollenin-bearing wall, the perine or perispore.

The other sporopollenin-containing elements associated with spores/pollen are, viscin threads, elaters, perispore like bands attached to the Equisetum spores, Ubisch bodies or orbicules. The aquatic flowering plants have abandoned the production of sporopollenin while members belonging to the family Lauraceae, Cannaceae, etc. manage to operate with very little sporopollenin in their exines.

Chemistry of Sporopollenin:

It is extremely difficult to characterize this highly inert compound, however, Zetzsche (1937) determined that the sporopollenin is an oxygenated hydrocarbon and contains hydroxyl and C-methyl groups and substantial level of unsaturation.

Shaw (1971) reinvestigated the structure of sporopollenin and confirmed the result of Zetzsche (1937). Shaw (1971) also obtained straight and branched chain monocarboxylic acids, which are characteristic breakdown products of fatty acids.

Besides this he found a mixture of phenolic acids. Shaw and Yeadon (1966) proposed that the sporopollenin is composed of a lipid fraction of 55-65%, consisting of molecules with a chain length of up to C₁₆, and a lignin fraction representing 10-15% of the total mass. The suggestion that sporopollenin was a mixed polymer of lipids and phenylpropanoid units were rapidly replaced by a chemical structure based on the polymerization of carotenoids and carotenoid esters.

Its empirical formula was worked out by Zetzsche (1937) in Lycopodium as C₉₀H₁₄₄O₂₇ It is extremely difficult to characterize the structural formula of sporopollenin as the techniques, which degrade it, produce simple sugars and other compounds, which obviously do not complement to the structure of the original molecule.

Moreover, the preparation techniques, like acetolysis and KOH lysis used to remove the other constituents of spores and pollen also change the sporopollenin, since when treated it has a marked tendency to pick up halogens, metallic ions, and other groups. Further acetolysis destroys some carbonaceous matter in the exine and introduces sulphur.

It is probably the most inert C-H-O organic compound that resists acetolysis, but degrades in strong oxidants like H₂O2₂, or CrO₃ and exhibits secondary fluorescence when stained with primuline. Shaw and Yeadon (1966), used the oxidizing agent ozone as the most valuable degradative reagent to decipher the monomers associated with sporopollenin.

The initial studies were devoted to Lycopodium clavatum and Pinus sylvestris because of their availability and high content of sporopollenin. The soluble organic compounds produced during the oxidation were found to consists of confusing range of mono- and di-carboxylic acids, both branched (C-methyl) and straight chain, with and without oxy (keto, hydroxyl) substituents.

There seemed to be no obvious pattern in these products, and one might therefore be justified in concluding that sporopollenin was derived from polymerization of a complex heterogenous conglomerate of plant chemical oddments linked into a vast formless structure.

Based on these studies and the work of Heslop-Harrison (1968) and Brooks (1969) it has been suggested that sporopollenin is a copolymer of β-carotene, and xanthophylls such as antheraxanthin, and fatty acids (Fig.2.6).

If this is true, the substance should have repeating units of an isoprenoid class. Further, the facts of Given (1984) that the straight chains predominates over isoprenoid structure in fossil spore walls, make it difficult to accept the Brooks and Shaw (1968) theory.

A further re-evaluation of its chemical structure has cast doubt on the role of carotenoids and favours the idea that sporopollenin, is a mixed polymer of phenylpropanoid and fatty acid derivatives. Prahl (1985) demonstrated that a potent inhibitor (norflurazon) of carotenoid biosynthesis had little effect on the formation of sporopollenin in Cucurbita pepo.

By applying ¹³C NMR spectroscopy it is seen that the sporopollenin is not a unique substance, but a series of related biopolymers derived from largely saturated precursors such as long chain fatty acids and oxygenated aromatic rings.

Traverse (1988) thus summarized the chemistry of sporopollenin as a highly inert C-H-O compound, probably of the carotenoid- terpenoid class. At present it is assumed that sporopollenin is a polymer consisting mainly of unbranched aliphatics with a variable amount of aromatics.

Its natural colour is pale yellow, but with thermal maturation, as the sporopollenin increases in rank, with loss of O and H and increasing percentage of C, the colour deepens through dark yellow, orange, reddish brown, finally to black. During this series the reflectance increases.

The agents of thermal maturation are temperature elevation plus time. The specific gravity is about 1.4 and the index of refraction is 1.48. It is sensitive to oxidation, but not so much as most of other organic matters in sediments. It is also sensitive to high pH over prolonged periods of time.

The staining and solubility differences between the ektexine and endexine indicate that the sporopollenin of these two layers differ chemically. If the wall is stained with basic fuchsin the ektexine, which is actually the sexine plus nexine 1, becomes heavily stained before the endexine or nexine 2.

Further, it has been shown that the ektexine of some pollen grains is soluble in hot 2-aminoethanol and several related substances while the endexine is left unaffected.

The measurement of the amount of sporopollenin in spores and pollen is usually achieved by acetolyzing them and assuming that only exine is left. In general, the more sporopollenin, the more resistant to decay, oxidation, etc. The distribution also makes a difference, the more sporopollenin concentrated in the outer part of the exine (ektexine or sexine), the durable is the exine (Table 2.1).

It is not affected by enzymes, so pollen and spore exine pass through most animal guts unchanged. It has been shown that the fern spores can even germinate in fair numbers after passing through the gut of a locust.

Moreover, it has been seen that some fungi can digest sporopollenin and can attack spores/pollen, apparently after deposition in sediments. Some bacteria can also accomplish this task presumably by their natural hydrolytic nature.

Production and Deposition of Sporopollenin:

It is presumed that some of the degradative products of the tapetal tissue undergoing senescence are utilized by the maturing pollen grains in the production of the sporopollenin. Based on light and electron microscopic observations it is assumed that tapetum is involved in the transfer of precursors of sporopollenin to the pollen wall.

Circumstantial evidences claim that the sporopollenin precursors are synthesized in the tapetal cytoplasm. Further evidences proclaim the accumulation of orbicules or Ubisch bodies with chemical properties of sporopollenin on the locular face of tapetal cells and their extrusion form the tapetum, followed by their alignment on the microspore wall where they are integrated into the developing exine.

The tapetum is thus in close and intimate association with the pollen grains maintaining a high degree of structural and functional organization until just prior to meiosis. It is thus clear that the tapetum must be having a considerable influence over the transport of material to the pollen grains.

Mepham and Lane (1970) have shown that bulk of the sporopollenin deposition occurs whilst the microspores are still invested with the callose layer. Once released from the tetrad, there is no further ektexine sporopollenin deposition, but synthesis of endexine sporopollenin continues over most of the inner surface of the pollen grain. They have further recorded narrow channels through the endexine as the young microspores expand after release from the tetrad.

The microspore cell membrane may protrude into these channels, and could make contact with the membrane of the tapetal protoplasts which infiltrate the inter bacular spaces during the later stages of development.

Such membranous contact could provide a route for the passage of sporopollenin precursors into the developing pollen grain. Figure 2.5 shows the possible sites of sporopollenin synthesis, polymerization, and deposition of sporopollenin as suggested by Heslop-Harrison and Dickinson (1969).

Sporopollenin is transported in a highly polymerized form from the tapetum to the exine. Electron micrographs indicate that isolated agglomerations of sporopollenin, surrounded by a unit membrane, called Ubisch bodies are found at a considerable distance from the microspore.

The endoplasmic reticulum widens to form pockets in which electron dense materials accumulate along the plasmalemma. These are the nuclei around which the spherical lamellae of sporopollenin granules, the orbicules will be deposited.

Once the pro-orbicular bodies pass through the plasmalemma they are rapidly coated with sporopollenin. The orbicules are transported across the tapetum cytoplasm into the locule of the pollen sac before being positioned on the microspore exine.

Sporopollenin seems to be arranged in bundles of anastomozing strands with an initial diameter of 80 to 150 A. Nearly four superimposed bundles of sporopollenin in the early wall separate to produce an exine composed of two shells, each having two superimposed bundles in cross section.

Later between meiosis and pollen mitosis, sporopollenin has a homogenous appearance. The ultrastructural details of the development of the amoeboid or periplasmodial and glandular or secretory tapetum type provide information as to how the tissues may be involved in the production of sporopollenin.

Function of Sporopollenin:

a) The oldest sporopolleninous acritarchs occur in Pre-Cambrian rocks, 1.2 to 1.4 billion years old. The presence of this unique plant polymer in such early sediments has important implications for investigations into the origin of life. In these early forms of life sporopollenin probably played the role of protector of the protoplasm against ultraviolet radiation.

The green algae were most probably responsible for the development of sporopollenin. In the course of evolutionary history it was introduced from them as a protective covering of the reproductive units in higher plants, with the prime function to act as a shield against oxidation and desiccation.

b) It is evident that the pollen exine has a basic function in the physiological complex of fertilization, since it has developed in almost all terrestrial plants irrespective whether they use wind or animals as pollination vector.

The sporopolleninous exine has been claimed to protect the protoplast from the rapid water loss. The barrier function of the exine for water, low molecular weight solutes and polymers is well established.

c) The resistant character of the sporopollenin to decay, oxidation, etc. is directly related to its amount and distribution. Higher concentration of sporopollenin and its greater deposition in the outer part of the pollen wall (ektexine or sexine) make the exine more durable, thus extending its viability.

b) Palaeopalynologically the significant property of sporopollenin is sporopollenin- bearing palynomorphs. Once delivered to sediment, they tend to stay there, though the contents and the other wall layers, of the palynomorphs are quickly lost.

Such an assemblage of varied palynomorphs by virtue of the sporopollenin help in the reconstruction of past vegetation, predicting climatic changes, understanding the evolution of plant life, and in the exploration of hydrocarbons (petroleum and coal) by using parameters like TAI (Thermal Maturation Index), and degree of carbonization.

However, oxidizing and highly alkaline environment, carbonization (= coalification), thermal maturation, as a result of relatively low temperature elevation over a long time, high temperature (due to volcanic intrusion) and recrystallization of minerals in the sediments degrade the sporopollenin, thus depriving us in understanding the evolution of plant life.

Conclusion:

Pollen, as the male gametophyte of plant has been widely studied on its developmental process, morphology, physiology and biochemistry for several decades. As an important feature of pollen structure and morphology, pollen wall has received great attention. It is known that pollen wall contains exine and intine.

The intine is mainly composed of cellulose and pectins, while the exine consists of sporopollenin, a biopolymer of extremely high chemical, physical and biological resistance. Pollen generally does not change its shape after long-term sediment under-ground, mainly due to the existence of pollen exine.

Although a lot of work has been done in approaching the formation pattern of pollen exine, the chemical structure and character of sporopollenin are not yet fully understood. It seems that the morphological unit of sporopollenin and the relationship for these units in pollen exine remain controversial.

The use of scanning electron microscopy (SEM), transmission electron microscopy (TEM) and treatment of hot 2-aminoethanol on the pollen exine structure led to the proposals of several different models of the exine structure.