Mirror Symmetry of Life

1. Introduction

Polarized environment, from the beginning of life on our planet, imposes on living organisms a necessity to read directions. Light and gravity are the two most important oriented signals that come from well-defined sources. The response to these primary polar signals allowed for development of the secondary, more sophisticated reactions to the network of other polar signals like gradients of chemical molecules or mechanical stresses. The signals of chemical and physical nature provide the extrinsic but also an intrinsic information for biological systems.

One of the basic features of the organism developing in polar environment is its axiality. This brings in consequence following possibilities: 1) multiplication of repetitive units of the body and their special alignment along the axis (segmentation, metamerism) and 2) deviations of structures from the axis to the left or to the right (development of L/R symmetry).

The main axis in the motionless plants, fastened to the ground, is mostly vertical, extending between the apical and basal poles. The animal main axis, regardless its position in the gravity field, connects the anterior and posterior poles. The axis formation starts at the very early stages of development. The identity of segments formed iteratively along the axis in both plants and animals is genetically controlled and their evolutionary multiplication creates a great potential for morphological and functional diversity through many useful modifications. This process is in a sense similar to the effects of gene duplication on the molecular level.

Subsequent emergence of L/R symmetry may be observed on all, hierarchically different levels of body organization. Some general principles, like minimum energy rule, are universal in nature leading to the identical solutions on all these levels. Good example represent spherical geodesic shapes. The structure of carbon allotrope - C₆₀ closed fullerene, is also present in coated endocytic vesicles reinforced by the clathrin cage [1], in regularly sculptured surface of pollen grains [2] and in a cellular pattern on the surface of the plant paraboloid apical meristem [3].

Other universal basic forms are chiral helices and spirals commonly observed on molecular, cellular and organismal levels. They are of particular interest here because of distinct mirror symmetry they have, which is the main focus of this chapter. Chirality of many macromolecules: nucleic acids, proteins or cellulose fibers [4], coiled coils of collagen, or such structures as tubulin cytoskeleton, thickenings of plant cell wall, plant tendrils, spiral snail shells or narwhal tusks are but a few examples. Not only structures but also some developmental processes may be chiral. The apical cell divisions in moss gametophores [5], cell cleavage in the embryos of snails [6, 7] or lateral organ initiation on plant shoot apical meristem (SAM) proceed clockwise (CW) or counterclockwise (CCW). Two interesting problems may be addressed while considering chiral structures in biological systems – mechanism of their formation and proportion between the two chiral configurations.

The aim of this chapter is to provide the readers with the overview of some examples of bio-chirality discovered over the years both in animals and plants. The stronger accent will be placed on the latter because they are less known and because they have always been in a focus of the author’s research. The mechanisms of many cases of mirror-symmetry presence in plants are yet to be elucidated.

2. Mirror symmetry on cellular level

Cell chirality or handedness is a newly discovered phenomenon, which nowadays is intensely studied, mostly in animal cells [7, 8, 9, 10]. It is manifested in the presence of chiral structures within the cells but also in the cell behavior that may lead to directional movements or assuming L or R orientation of cell alignment. In animals it affects organs laterality [10], in plants results in development of spiral, helical or wavy patterns [3, 11, 12, 13, 14, 15].

During primary axis formation on the cellular level the polarity of the cell is manifested in an uneven distribution of receptors, ion channels and hormone carriers on plasma membrane, and internally in the ion currents and cytoskeletal fibers parallel to the developing axis but also in the polar distribution of ultrastructural components like cell organelles or nutrients. All these sophisticated processes have been investigated mainly in plant egg cells or fucoid zygotes [16, 17, 18] and animal oocytes [19, 20]. However, even in the integrated system of multicellular organism, singular cell polarity is often a case. In animal body the epithelial cells of intestines constituting a planar 2D barrier, have their polarity unified. It is manifested in nonrandom distribution of glucose transporters which facilitates the oriented transepithelial sugar transport [21]. In plants the polar distribution of the auxin influx (AUX) and efflux (PIN) carriers in plasma membrane results in polar transport of the hormone between the cells [22]. In L1 layer of SAM auxin is transported acropetally, whereas inside of the plant body, in the provascular tissues and later in cambium, the hormone transport is basipetal. Change in the distribution of carriers and thus of the cell polarity redirects the transport, often affecting the directions of plant organ growth [23]. This, for instance, has been noted in gravitropic response of the roots [24].

Many elements of the cell ultrastructure are spectacularly chiral. In some green algae exemplified by multicellular filamentous Spirogyraor unicellular Spirotaeniaand Chlamydomonas spirogyroidesthe chloroplasts are of considerable length, flat and ribbon-like. They assume helical course in the cortical cytoplasm of the cell. Not much is known about the chiral configurations of their coiling. Images available in various data bases suggest that in Spirogyraboth configurations may be present in different filaments [25], in different cells of the same filament or even within the same cell [26]. However, the error resulting from improper focusing during microscopic observations cannot be excluded. The sample taken for analysis from the aquarium of the Botanical Garden of Wrocław showed under light microscope hundreds of cells of the same S configuration of chloroplasts coiling from the right to the left ( Figure 1 ). Mechanisms by which the configuration is regulated remain undiscovered.

Another clearly chiral component of the cell is basal body. In eukaryotic, plant and animal cells some identical structures bear different names although they look the same. Two centrioles of the centrosome, basal bodies or kinetosomes in the motile or ciliary epithelial cells have the same architecture. Composed of 9 triplets of microtubules (MT), overlapping on all available images either CW or CCW, they resemble a pinwheel toy. It is unclear, however, whether both configurations, being a mirror-image of one another, are indeed present in all different types of the cells. Transmission electron micrographs (TEM) of tangentially sectioned cell surface show that in a particular cell all basal bodies underlying cilia are of the same chirality [27]. However, unless it is clearly stated like in [28], it is not known whether basal body is seen on TEM from the surface of the cell or from its inside. This is the reason for chiral configurations of basal bodies being uncertain. The image of Paramaecium micronucleatumby Dennis Kunkel [27] shows CCW overlapping triplets, whereas in Paramecium tetraurelia[29] the triplets overlap CW.

In flagellar apparatus of Chlamydomonasor Acrosiphoniagamete both chiral configurations of basal bodies are present on the same electron micrograph [30, 31] but in these cases it is certainly an effect of opposite orientation of flagella and their two basal bodies facing each other horizontally. The bodies are in fact of the same chirality. This case shows again how careful one must be analyzing and interpreting the examples of mirror-symmetry of the chiral ultrastructural components of the cell on TEM images, that can be easily flipped and show the same structure either from above or from the bottom side. There is one additional aspect of mirror symmetry presence in the locomotion apparatus of green algae. The flagellar roots of the two basal bodies are slightly rotated one with respect to the other – CW in representatives of Chlorophyceae and CCW in Ulvophyceae [30, 32, 33]. This finding, among the others, was the foundation of the profound revision of green algae taxonomy [30, 33].

The process of cilia beating is chiral. Both ciliates, motile spermatozoids and stationary epithelial cells readily change the direction of cilia movement either to navigate or alter the current of surrounding fluids [34]. Interestingly, the latter has been employed by unicellular Stentor rosei, to avoid, in quite deliberate and calculated manner, the irritating particles experimentally added to the medium [35]. The cell, however, is not always in full control over the cilia beating. The doublets of some ciliates, having notably the same chirality of basal bodies, show that in the form being a mirror image of the typical one, food particles are expelled from the oral apparatus instead of being directed towards it [28]. Typical chiral form of Paramaeciumswims forward employing leftward rotation. Stressful conditions like low temperature or heavy metals force the ciliate to spin in opposite direction [36].

Among fibrillar elements constituting cytoskeleton, a tensegral structure of eukaryotic cytoplasm it is actin microfilament (MF) that is chiral. Mammalian cells exhibit specific, actin dependent L/R asymmetry which is different in normal and cancerous cells and changes when inhibitors of actin function are applied [37]. In bacteria changeable chirality of actin homologs MreB has impact on their growth and cell shape [38]. Actin is also responsible for directional, chiral movement of cytoplasm in the cells of charophytes [39]. Another important component of cytoskeleton, MTs. per seare not chiral. However, their arrangement in the cortical cytoplasm, beneath plasma membrane, is often oriented in plant cells ( Figure 2 ). The elongating cells in the axial organs of the model plant Arabidopsis thalianaapparently have the chirality of their cortical cytoskeleton genetically controlled. In spiral 1and spiral 2mutants the cortical MTs, watched from the cell surface, form an ascending S helix, whereas in leftymutant the helix is Z. The mutations lead to abnormal growth of the plant axial organs which, strangely enough, become twisted oppositely to the configuration of MT helix in their cells – Z in the spiral, S in the leftymutants [40, 41, 42].

Configuration of cortical cytoskeleton, below plasma membrane, in some differentiating plant cells may change with time causing the development of interlocked pattern of the cellulose microfibrils deposited in the secondary wall [43]. Both chiral systems: intracellular and extracellular, parallel each other in consecutive stages of the secondary cell wall formation. The cycle of changes in microfibrils orientation always starts from the ascending S helix in S1 layer of the secondary wall (watched from the outside of the cell), it alters to Z in the S2 layer and returns to S helix in the S3 layer. No exception from this rule has been found even though, at least theoretically, the opposite sequence of changes in microfibrils orientation is possible. Moreover, the cycle of changes in the chirality of microfibrillar helix appears to be independent of the overall orientation of the differentiating cell within figured wood, which also exhibits L/R symmetry [44]. This astounding regularity points to existence of yet undiscovered mechanism that must precisely regulate the phases sequence in the full cycle of changes in the cortical MTs orientation. Must be also independent of the other, hypothetical one, which controls L/R symmetry of cambial cellular events such as oriented anticlinal divisions and intrusive growth. This assumption is supported even further by the S helix of the secondary wall thickenings in differentiating protoxylem. The first, S1 deposit of this wall, not yet completely covering the primary wall surface prevents the cells, exposed to mechanical stress caused by longitudinal expansion of growing shoot, from breaking ( Figure 2 ).

The discovery of cell intrinsic chirality resulted from the fundamental question how the laterality of organs within the animal body is accomplished. Over the years much attention has been paid to this phenomenon and its connection with the development of L/R symmetry of the whole multicellular organism. It was found that blood neutrophiles polarity, defined by position of centrosomes with regard to the cell nucleus, makes them capable of directional movements in absence of polar external signals. This property disappears after application of drugs affecting MT function [45].

The model invertebrate organism Drosophila melanogasterprovided evidence that myosin encoding gene mutation switches cell chirality and results in the development of situs inversusphenotype of the hindguts or genitalia [7, 8, 10, 46]. In vertebrates the development of typical L/R asymmetry from anteceding state of embryo bilateral symmetry is generated by various mechanisms. One of them is based on function of axonemal dynein. This motor-protein is responsible for appropriate beating of cilia in nodal epithelial cells, causing the directed ion current. Defects in the gene structure encoding for the dynein results in random selection of heart position in mouse embryo [47]. The involvement of C kinase signaling pathway in the reversal of cells chirality leading to mirrored position of heart was recently shown in chicken embryos [48].

All the above studies show how intrinsic cell polarity may be translated onto the higher level of multicellular organism organization in animals. Very little though is known about L/R symmetry regulation in multicellular plant organisms.

3. Chiral processes and structures in multicellular organisms

The axiality of multicellular organism is similar to the polarity of a single cell but on hierarchically higher level of organization. The first evolutionarily step towards axiality of the integrated biological system composed of many cells represent 1D filaments of cyanobacteria, eukaryotic algae and fungi or moss protonemata. Higher plants maintain this ancestral condition as a kind of atavistic trait at the embryonic stage of their development - linear suspensor, which originates from the basal cell of already polarized and divided zygote, transports nutrients to the 3D globular embryo, developing from the apical cell [18]. Many forms of animals with such model organisms as tiny worm Coenorhabditis elegans[49] or fruit fly Drosophila melanogasterand finally ourselves, exhibit axiality, metamerism and L/R symmetry.

In this section the short survey of the most interesting cases of mirror symmetry in multicellular plants and animals will be made and the mechanisms that stay behind them will be discussed.

3.1 Changing chirality in the thalli of charophytes

Architecture of these green algae resembles that of the horsetails. The thallus of Charais composed of the giant internodal cells typically enveloped by the sheet of cortical cells, which take an origin from the adjacent nodal cells. From the nodes the 1-st order branchlets grow out horizontally, on which the reproductive structures are positioned: ovaloid oogonia and spherical antheridia. They may be treated as the 2-nd order axial outgrowths of the branching thallus. The peculiar, to date unexplained transition takes place from the Z orientation of enveloping cortical cells in the main axis of the thallus, through their mostly parallel alignment in the branchlets, to the S orientation in oogonia ( Figure 3 ). The developmental sequence of these changes in the chiral structure of extant Charaspecies is always the same, although gyrogonites (fossilized oogonia) of charophytes show that in the late Devonian period the right-handed (Z) oogonia were also present. They belonged to the charophyte family Trochiliscaceae and got extinct with the onset of Mesozoic era [50, 51, 52]. We will probably never know if they had the same sequence of chiral changes, but in reverso, like the extant Charabranching thalli.

The cortical cells of Charaparallel orientation of the cytoplasmic streaming in the central internodal cell they envelop. How do they read this direction? How is this information translated from the interface between ecto- and endoplasm, where the cellular engine of cyclosis is located [39, 53, 54], to the surface of the cell wall, on which the enveloping cells slide? Finally, how and why does it change in the Charabranching system, from the right in the main axis to the left in oogonia? Is actin – motor protein involved? Actin microfilaments must then have orientation of their alignment determined by the position of the cell in branching thallus. How? These problems remain unresolved.

3.2 Oriented cell divisions in apical cells of mosses and ferns

The control over orientation of the cell division plane is of particular importance in plant tissues. Plant protoplasts are “imprisoned” within the boxes of their cell walls. They cannot migrate as freely as do the animal cells during embryonic stages of ontogenetic development. Morphogenesis of plants relies entirely on the properly oriented cell divisions.

In the simple multicellular filaments of green algae we encounter for the first time the manifestation of L/R symmetry. The plane of cell divisions may be inclined relative to the filament axis either to the left or to the right as exemplified by the filamentous green alga Coleochaete nitellarum[55]. Also in planar (2D) gametophytes of ferns the pyramidal apical cell (AC) with rectangular base divides alternately to the left and to the right in a regular sequence ( Figure 4 ).

Precision in controlling chiral configuration of cell divisions is even more striking in 3D leafy gametophores of mosses. Their tetrahedral AC, watched from its triangular base, divides either CW or CCW and this direction is randomly established in the development of the gametophore main axis. However, it is not so in the case of its lateral branches – the chirality of their AC is always opposite to that of the supporting axis [5]. It is possible that this antidromic correlation triggers the horizontal gradient of some putative signals vertically transported from the neighboring leaves of gametophore ( Figure 5 ). Two genes of the model moss Physcomitrella patenswere identified to be engaged in a process of cell divisions in leafy gametophore: PpTONE1controlling intracellular organization of MT cytoskeleton and PpNOG1assuring proper development of AC [56, 57].

3.3 “Music of trees”

Anticlinal, pseudotransverse divisions of elongated stem cells in cambium, cylindrical meristem located in trees between the outer bark and the inner secondary xylem, are chiral. Their partitions, while watched from outside of the tree, are inclined to the right (Z divisions) or to the left (S divisions). Cambium therefore is a plant tissue that exhibits clear L/R symmetry. Also subsequent growth of the cells shortened by the divisions is oriented. Cambial cellular events are not randomly distributed over the surface of the meristem but orderly segregated into domains of opposite chirality. The S and Z domains alternate along the vertical axis of the cambial cylinder [58, 59]. This leads to emergence of structural waviness within which the cells assume alternately the opposite S and Z orientation. Because cambial structure is replicated every year in the annual wood increment, the history of all these developmental changes is recorded in the wood and may be extracted for the periods equaling the age of a tree. The domain pattern and resulting structural waviness are propagated vertically in cambium thus the cells in a particular location undergo the cycle of inclination change ( Figure 6 ). This barely known biological rhythm is the longest in nature. Its period approximates 20 years although in some cases may be shorter. Theoretical model predicts that propagation of cell oscillations associated with the domain pattern motion may lead to development of the spiral grain in a tree trunk [14]. Handedness of the spiral grain should depend, according to the model, on the nature of the wave front i.e. the direction of the first change in stem cells inclination during the initial period ( Figure 7 ).

Neither the molecular mechanisms, nor the nature of domain positional information for dividing and growing cells have been elucidated so far. The first suspect is the polar auxin transport changing directions due to redistribution of the hormone carriers in plasma membrane of cambial stem cells. The intrinsic cell chirality resulting from unknown nature of the intracellular oscillator cannot be excluded. In one tree more than one domain pattern may be present – they usually differ in the domain size and propagation velocity. According to the hypothesis put forward by their discoverer, the domain patterns result from morphogenetic waves traveling in the tissue and capable of superposition [59]. This means that the trees play silent music, the beauty of which is mostly unknown even to the scientists.

3.5 Aestivation

The petal folding in a flower bud, in most of the flowering plants, is clearly chiral. Petals overlap either CCW or CW and this chiral configuration is often later maintained in fully developed flower ( Figure 8 ). The direction of petals folding may be, like in the case of circumnutation, the species specific trait.For instance, Anagallis arvensispetals always twist CCW, whereas in Hawaiian plumerias they do it otherwise. Common European weed Malva neglectain turn is heterochiral, capable of producing CW and CCW buds on the same individual plant.

3.6 Phyllotaxis

Among best known chiral phenomena and investigated since the ancient times [66] is helical phyllotaxis – the regular distribution of lateral organs such as leaves or flowers on a plant shoot. Their consecutive primordia, circumferentially equidistant, emerge on the vertically growing shoot apex in the regular intervals. The primordia may be connected with an imaginary line called ontogenetic helix. The helix S or Z configuration depends on whether the process of primordia initiation proceeds CW or CCW. The plantlets growing from seeds have this configuration established at random in the main axis. It is not so, however, in the axes of lateral branches. Their ontogenetic helix may be either concordant (a homodromy case) or discordant (an antidromy case) with that of the supporting axis. It has been found, that even when both phyllotactic correlations occur with the same frequency [67] the supporting axis and the laterals may have the same chirality of vascular sympodia - elements of the internal transport system strongly related to phyllotaxis ( Figure 9 ).

The sympodia follow the course of one set of superficial secondary helices - phyllotactic prastichies. Two sets of parastichies running in opposite directions constitute a phyllotactic lattice. This is why even when ontogenetic helices in two axes making up one branching unit are discordant, the axes still may be concordant on the level of their vasculature. The numbers of parastichies in the sets of opposite chiral configuration belong to the mathematical series, the quality of which is associated with the size of circumferential distance between successive primordia. This distance, usually given in an angular measure, is known as divergence angle. The most common is the main Fibonacci series (1,1,2,3,5,8,13…) present in the system with the divergence angle approximating 137,5 degrees or Lucas series (1,3,4,7,11 …) with the angle close to 99,5 degrees. There are also many other divergencies and phyllotactic patterns [68].

Asymmetry of phyllotactic lattice with regard to the shoot axis is most probably responsible for the peculiar twirling of needles frequently seen on the top of coniferous shoot ( Figure 9 ). The chirality of these twirls results likely from the growing shoot torsion and is rather related to the orientation of vascular sympodia than to the chiral configuration of ontogenetic helix.

Regularity of primordia initiation resembles crystal growth. The plant apical meristem where the primordia are tightly packed may be called by licentia poetica, a living crystal [69] ( Figure 10 ). The similarity has been strengthened by the discovery that in phyllotactic lattices dislocations occur [68, 69, 70]. Single dislocation often changes not only a quality of the pattern but, most importantly, the chirality of ontogenetic helix ( Figure 11 ).

4. Conclusion

There is no one universal mechanism that stays behind the mirror symmetry of life. The frequency of both chiral configurations is not the same in different biological systems. However, as it has been discussed here, it was not always assessed carefully enough by investigators. Spirogyracase is uncertain. The narwhal tusks are probably always S-helical. On the contrary the shell coiling in most of the snails is of Z type. In many systems the chiral configuration of structures or processes is strictly controlled, in many others the control is loose or absent, which results in equal frequency of both S and Z forms.

In light of the newly discovered intrinsic cell chirality in animal cells we have now a great perspective of disentangling the ultracellular and molecular basis for the dynamic wavy and spiral patterns developing in cambia of trees - one of the most intriguing and least known biological rhythms. Discovered and thoroughly characterized by Hejnowicz and his followers in the last decades of past century it still remains a great mystery. Neither the nature of specific positional signals coming from the dynamic morphogenetic field to the cambial stem cells nor the mechanism of their response i.e. S or Z oriented cell divisions on the cylindrical surface of this embryonic tissue, have been elucidated. The tissue is also intriguing because of its structure. It may be compared to that of the liquid crystals – the elongated cambial stem cells may be aligned in the regular horizontal tiers, like the molecules in the smectic phase of the liquid crystal, or irregularly but parallel to the vertical axis, like in the nematic phase. The oscillating cambial cells taken together with their derivatives, continuously rotated in the successive wood layers, resemble the third, cholesteric phase of the liquid crystal [76].

Biomechanics of structures based on the possibility of changing chiral configurations, clearly the adaptive trait, cannot be underestimated. The resulting interlocked systems provide high resistance to mechanical stress. Interlocked are the cellulose microfibrils in the successive layers of the secondary cell wall in a single cell and, on the macroscale, the oppositely oriented wood fibers in the packets of consecutive wood increments of such giants as mahogany or camphor trees. Also the system of cortical resin canals in the young coniferous shoots, which runs oppositely to oriented vascular sympodia strengthens the axis mechanically.

These examples together with the crystalline character of phyllotactic patterns and cambial cells arrangements bring us back to already mentioned, at the beginning of this chapter, universality of some solutions based last but not least on the presence of mirror symmetry of life.

Acknowledgments

The author expresses sincere thanks to her long life associates in the Department of Plant Developmental Biology at the University of Wroclaw, Poland for their continuous inspiration and support; especially to Dr. Katarzyna Sokołowska, who helped here with Figure 5 , Dr. Alicja Banasiak, expert on plant cell walls and polar auxin transport and to Magdalena Turzańska (MSc), an excellent bryologist, for our endless discussions on the hidden beauty of a small world she immortalizes on her artistic microphotographs.