[307] Only that algal group commonly believed ancestral to land plants produces lignin from monomeric precursors. The lignin content of common plants can be reduced by gravity compensation and increased by mechanical load. Evolutionary opportunism?

At some point in early Paleozoic time-the Upper Ordovician perhaps-vascular plants began their occupation of the wetlands interfacing both terrestrial and marine environments. Whether this movement was a bona fide invasion driven by increasing population pressures in the marine littoral, an accident of gradual subsidence, or the aftermath of a period of exceptional tides will probably never be known, but the impact on the terrestrial landscape and on the course of subsequent evolution is unmistakable.

Despite their juxtaposition, these early populations, save for thalloid forms appressed closely to rocks, can hardly be considered to occupy a safe transition zone. The early Psilophytean derivatives, with their emergent vertical, branched axes, left behind an existence in the comfortable protective sea and immediately faced problems of support, water supply, gas exchange, and radiation exposure. To be sure, solutions calling for minimal adaptation exist within the time dimensions of tidal periodicity, but these must have limited the deployment of terrestrial life forms to the strand and coastal marsh-the continental fringe-with no guarantee of permanence.

We are here concerned with those forms able to cope with a more or less modern atmosphere, strong sunlight, little water, and unrelieved gravity, and with the ways in which they may have differed constitutionally from their Chlorophycean ancestors. If we neglect the host of minor and inconsistent variations seen among modern green algae (and the more organized [308] Charophytes) and their most primitive vascular descendants, one of the most consistent differences resides in the abundance of phenolics, especially lignins, nonnitrogenous polymers which may comprise 10, 30, 50% or more of the total dry matter in trees (Brauns, 1952; Wise, 1952) and are significant constituents, with important exceptions, even of small herbaceous vascular plants. In contrast, the phenolics of the green algae consist principally of tyrosine and related aromatic amino acids, which make up a small to minute percentage of dry matter, almost completely in proteins and peptides.

To what extent, then can lignins account biophysically and biochemically for the adaptive processes involved in the emergence of the vascular land plant? And how is this process of lignification programmed and regulated ?

Can the antiquity of the lignins be verified? Spectrochemical and chemical analysis of Devonian and Pennsylvanian fossils verifies the presence of phenyl methyl ethers, characteristic of ligning; and in the case of Calamites and Lepidodendron, extractable lignin-like fractions have been obtained in small amounts (Manskaya, 1959; Siegel et al., 1958; Siegel, 1968). More recent specimens (Pliocene) contain large amounts of lignins with reactive aldehyde groups as well as methyl ethers (Siegel,1968).

In other respects, answers to questions relating to the phylogeny and adaptive significance of the lignins must depend on experiments and observations using contemporary plant material. The remainder of this paper will summarize a series of such observations.

Observation: Conversion of orthosubstituted para-hydroxycinnamic acid to lignin requires the enzyme peroxidase (Higuchi, 1959; Siegel, 1968; Siegel et al., 1960). Although forms of this enzyme occur and even abound in many algae (table 1), only those present in the Chlorophyta can utilize the lignin precursor as a substrate (Siegel and Siegel, 1970).

Significance: Only that algal group commonly accepted to be ancestral to land plants can produce lignin from monomeric precursors.

Observation: When peroxidase-loaded fibers are incubated with lignin precursors, polymer is deposited on polysaccharides but not on proteins. And less-crystalline, lower-molecular-weight polysaccharides are more "efficient" by one or two orders of magnitude (table 2).

Significance: Fiber models show that the polymerization process is selective of the plant cell surface, but that secondary-wall polysaccharides (pectin and equivalents) are favored. This corresponds to established histochemistry (Siegel, 1956, 1957,1959; Wardrop and Bland, 1959).

Observation: All terrestrial vascular plants, angiosperms (and gymnosperms), ferns, horsetails, etc., contain lignin (table 3), whereas aquatic and marine vascular forms and ordinary nonvascular plants do not (Siegel et al., 1972). Nevertheless, all plants tested that had Chlorophycean affinities can....

....synthesize lignin from p-hydroxycinnamic acid -type monomers such as caffeic acid or coniferaldehyde. Excluded are the red and brown algae, blue-green forms and the fungi (with rare exceptions only among wood-rotting types) .

Significance: The inability to realize a potential for lignification means that (a) it never developed or (b) it is not now operational. The aquatic and marine vascular forms are generally regarded as "cetaceans" of the plant kingdom with a fully developed land ancestry (Siegel, 1979) followed by loss or "shutdown" of the required capabilities for continued existence out of water (option (b)).

Among the nonvascular forms in aquatic-marine habitats, the nongreen algae are assigned to option (a), but the status of the green algae is more conjectural. Originally, the Chlorophyta were assumed to be in option (a), but this may be the modern condition. Lignified fibers, lacking resiliency, confer brittleness on pliant rubbery algal tissues, and lignified algae anchored along rocky coastlines may well have disintegrated under wave impact. Conceivably, the capability for lignification only appeared among evolving wetlands or intertidal populations in latent (repressed?) form and was subsequently activated.

[311] Is there then any evidence for the presence of the requisite information for lignification in latent and/or facultative form?

Observation: In mosses, the sporophyte (2N) generation contains semilignified structures whose function is tied to its hydrophobic character. The common 1-10 cm gametophyte (1N) axis contains less than 0.3% lignin, if any (fig. 1). Gametophytes of 40-60 cm may contain 5-6% lignin, and the axes of the giant New Zealand mosses, up to 100 cm in height, contain 10-12% lignin (Siegel et al., 1972).

Significance: Even the smallest moss contains genetic information for lignification, but only those extended sufficiently above the surface plane encounter mechanical stresses sufficient to induce its redeployment into the upright axis.

[312] Observation: In addition to mosses, dwarf, ordinary, and giant ecotypes of common plantain and horsetail vary in total lignification with axial height (fig. 1). Even within the axis of a semiwoody plant, the lignin content from tip to base is closely related to mechanical loading (pressure of the axis on itself; fig. 2).

Significance: Either the original (nonvascular) ability to synthesize lignin was not size-related but became so in the mosses as their axes were extended upward, or the mosses and their relatives are degenerate forms of vascular origin. In vascular plants, lignin is largely facultative and mechanical load-dependent. It may thus be a function of the gravity-mass interaction

Observation: By the use of clinostat, flotation, and centrifuge tech niques (Chen et al., 1980; Siegel, 1968; Siegel et al., 1972, 1977; Waber et al.,1975), the lignin content of common plants can be reduced under conditions of gravity compensation and increased with mechanical load (table 4). Of particular note is the response of Elodea, an angiosperm returned to the aquatic habitat. We have shown that Elodea, without growth, can be induced to form lignin from endogenous sources whiie on the centrifuge for 6 days (Chen et al., 1980).

Significance. The inference drawn (earlier) from size-lignin relations that lignification is a function of gravitational load is here confirmed usingg as the variable. However, the original conclusion (Siegel, 1953) that Elodea.....

....and related aquatic or marine forms had lost the capacity for monomer, but not polymer, synthesis, is refuted. The alternative is gravity-induced derepression on induction of a long-dormant program for monomer sy nthesis.

We assume that the gravitational "switch" operates on phenylalanine/ tyrosine -deaminases, which convert the aromatic amino acids into cinnamic and p-coumaric acids, respectively (Brown, 1964; Conn, 1964; Neish, 1964). These in turn, after relatively minor modifications, become monomers.

Observations: Withholding water from air-grown cucumber seedlings enhances lignification (table 5). Similar effects can be obtained with watergrown plants in a saline medium. An O₂ deficiency has the opposite effect. Both cucumber and bean seedlings grown in atmospheres of 5% O₂ have about half the normal lignin content (Siegel, 1953).

Significance: Lignins are hydrophobic, resinous substances and have long been recognized as waterproofing agents for vascular and epidermal cells. The lignins thus overlap with the polyester epidermal covering known as "cutin," not only in the waterproofing role, but often in the outer walls where the spectrochemical properties of the lignins, strong extinction of near ultraviolet radiation, can be expressed (Eglinton and Hamilton, 1967). Their function in smaller moss sporophytes, as noted above, is also related to this hydrophobic character. Reductions either in bulk water supply or in water activity apparently comprise or generate a signal which in turn promotes lignin synthesis.

The role of O₂ in this system is simple and direct. It is needed for synthesis of specific monomers from phenylalanine or tyrosine, but its principal.....

....role is in monomer dehydrogenation to the lignin polymer (Siegel et al 1972). The system is first order in O₂ pressure; thus any variations in oxygen concentrations will be reflected directly in rates of peroxidation

The differences between modern Chlorophyta and vascular land plants cannot be explained in full on the basis of lignification alone. Nevertheless, the data pomt to the emergence of the primitive land populations into an oxygen-rich terrestrial world where the need for mechanical support, water conservation, and, to some degree, radiation protection could be met by a smgle aerobic biochemical process connected to essential aromatic amino acids hkely to be found in every cell. If there is such a thing as evolutionary opportunism, this must be a good example.

This work was supported in part by National Aeronautics and Space Admmistration grants NASW-767 and NGR 12-001-053 and Contracts NAS2-6624 and NAS2-8687.

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