Complex reproductive secretions occur in all extant gymnosperm lineages: a proteomic survey of gymnosperm pollination drops.

KEY MESSAGE: Complex protein-containing reproductive secretions are a conserved trait amongst all extant gymnosperms; the pollination drops of most groups include carbohydrate-modifying enzymes and defence proteins. Pollination drops are aqueous secretions that receive pollen and transport it to the ovule interior in gymnosperms (Coniferales, Cycadales, Ginkgoales, Gnetales). Proteins are well established as components of pollination drops in conifers (Coniferales) and Ephedra spp. (Gnetales), but it is unknown whether proteins are also present in the pollination drops of cycads (Cycadales), Ginkgo (Ginkgoales), Gnetum (Gnetales), or in the pollination drops produced by sterile ovules occurring on pollen plants in the Gnetales. We used liquid chromatography-tandem mass spectrometry followed by database-derived protein identification to conduct proteomic surveys of pollination drops collected from: Ceratozamia hildae, Zamia furfuracea and Cycas rumphii (Cycadales); Ginkgo biloba (Ginkgoales); Gnetum gnemon and Welwitschia mirabilis, including pollination drops from both microsporangiate and ovulate plants (Gnetales). We identified proteins in all samples: C. hildae (61), Z. furfuracea (40), C. rumphii (9), G. biloba (57), G. gnemon ovulate (17) and sterile ovules from microsporangiate plants (25) and W. mirabilis fertile ovules (1) and sterile ovules from microsporangiate plants (138). Proteins involved in defence and carbohydrate modification occurred in the drops of most groups, indicating conserved functions for proteins in pollination drops. Our study demonstrates that all extant gymnosperm groups produce complex reproductive secretions containing proteins, an ancient trait that likely contributed to the evolutionary success of seed plants.

Cracking the omega code: hydraulic architecture of the cycad leaf axis.

Background and Aims: The leaf axis of members of the order Cycadales ('cycads') has long been recognized by its configuration of independent vascular bundles that, in transverse section, resemble the Greek letter omega (hence the 'omega pattern'). This provides a useful diagnostic character for the order, especially when applied to paleobotany. The function of this pattern has never been elucidated. Here we provide a three-dimensional analysis and explain the pattern in terms of the hydraulic architecture of the pinnately compound cycad leaf. Methods: The genus Cycas was used as a simple model, because each leaflet is supplied by a single vascular bundle. Sequential sectioning was conducted throughout the leaf axis and photographed with a digital camera. Photographs were registered and converted to a cinematic format, which provided an objective method of analysis. Key Results: The omega pattern in the petiole can be sub-divided into three vascular components, an abaxial 'circle', a central 'column' and two adaxial 'wings', the last being the only direct source of vascular supply to the leaflets. Each leaflet is supplied by a vascular bundle that has divided or migrated directly from the closest wing bundle. There is neither multiplication nor anastomoses of vascular bundles in the other two components. Thus, as one proceeds from base to apex along the leaf axis, the number of vascular bundles in circle and column components is reduced distally by their uniform migration throughout all components. Consequently, the distal leaflets are irrigated by the more abaxial bundles, guaranteeing uniform water supply along the length of the axis. Conclusions: The omega pattern exemplifies one of the many solutions plants have achieved in supplying distal appendages of an axis with a uniform water supply. Our method presents a model that can be applied to other genera of cycads with more complex vascular organization.

Distribution of gelatinous fibers in seedling roots of living cycads.

UNLABELLED: • PREMISE OF THE STUDY: The presence of gelatinous (tension) fibers (GFs) in the roots of two extant cycadales (Cycas and Zamia) in a recent publication raises interesting issues of GF distribution in seed plants. An immediate question that arises from this discovery is whether GFs occur consistently in the radicle of all extant cycad genera and therefore might have a similar role in root contraction. We present results of a survey of nursery-grown material of all 10 genera.• METHODS: We sequentially sectioned seedling root material and used simple staining and histochemical methods to follow anatomical changes along the radicle of all 10 genera.• KEY RESULTS: We found GFs in nine genera; Stangeria appears to be the only genus without them. In all genera, there is a wide variation in the number of GFs and also variation in the development of thickened, fleshy roots. "Tertiary expansion" is a useful term to describe late cell division and enlargement of both primary and secondary parenchyma, the latter produced by the vascular cambium. Certain other histological features can be diagnostically useful at the generic level.• CONCLUSIONS: The functional interpretation of GFs as being wholly responsible for apparent tissue contraction is now somewhat compromised, especially as distortion of tracheary elements by changes in dimensions of parenchyma cells can falsely suggest root contraction when it may not occur. These preliminary results point the way to a more precise investigation of study material grown in more uniform environments using advanced technological methods.

Root contraction in Cycas and Zamia (Cycadales) determined by gelatinous fibers.

UNLABELLED: • PREMISE OF THE STUDY: Reaction wood (RW) in seed plants can induce late and usually secondary changes in organ orientation. Conifers produce compression wood (CW), generated by compression tracheids, which generate a push force. Angiosperms produce tension wood (TW), generated by tension wood fibers (TWF) often described as "gelatinous fibers," which exert a pull force. Usually RW is produced eccentrically, but it can occur concentrically, as in aerial roots of Ficus. However, gymnosperms can produce gelatinous fibers (tension fibers, TF), as in cortical and secondary phloem tissues (Gnetum). TFs are therefore limited neither to wood, xylem, nor angiosperms. Here we demonstrate that TFs in secondary phloem are involved in contraction of roots of cycads and compare them with TFs of Ficus.• METHODS: We sectioned root material of cycads at various stages of seedling development using simple staining and histochemical procedures to follow the course of secondary phloem development. Aerial roots of Ficus were compared with the cycad root material.• KEY RESULTS: Tension fibers (gelatinous fibers) occur extensively and continuously in the secondary phloem in roots that undergo contraction. Older tissues, but notably the xylem, become distorted by contraction. TFs in cycads correspond in cell wall features to TFs that occur in Ficus, but do not occur in secondary xylem. The individual fibers visibly contract.• CONCLUSIONS: Tissue contraction in Cycas and Zamia corresponds to that found in angiosperms and Gnetum and further broadens the scope of the activity of tension tissues. This finding possibly indicates that gelatinous fibers originated at a very early period of seed plant evolution.

Palms do not undergo secondary stem lengthening: a response to Renninger and Phillips (American Journal of Botany 99: 607-613).

Woody stems that have completed some maturation of metaxylem elements should not be capable of further axial extension ("secondary stem lengthening"). However, this mechanism has been claimed by Renninger and Phillips (American Journal of Botany 99: 607-613) to be a feature of the palm Iriartea deltoidea. In response, we describe structural features of palm stems based on extensive known features of their anatomy and development. In addition to the inability of metaxylem vessels to extend after they are mature, fully differentiated fibers of the vascular bundle sheath, which would exist at the time of proposed stem elongation would not be capable of belated extension. "Vessel spirals" claimed by these authors to be capable of stretching to accommodate secondary stem lengthening does not refer to well-established features of the course of vascular bundles. The approach adopted by Renninger and Phillips simply measures stems of different sizes as an implied developmental series. Consequently, results do not take into account changes in the development of the palm stem as it ages. The existence of secondary stem lengthening in the palm Iriartea deltoidea, something never before observed in any tree, cannot occur because it would indeed disrupt mature metaxylem vessels and would also require the secondary extension of mature lignified fibers.

Cell longevity and sustained primary growth in palm stems.

Longevity, or organismal life span, is determined largely by the period over which constituent cells can function metabolically. Plants, with modular organization (the ability continually to develop new organs and tissues) differ from animals, with unitary organization (a fixed body plan), and this difference is reflected in their respective life spans, potentially much longer in plants than animals. We draw attention to the observation that palm trees, as a group of monocotyledons without secondary growth comparable to that of lignophytes (plants with secondary growth from a bifacial cambium), retain by means of sustained primary growth living cells in their trunks throughout their organismal life span. Does this make palms the longest-lived trees because they can grow as individuals for several centuries? No conventional lignophyte retains living metabolically active differentiated cell types in its trunk for this length of time, even though the tree as a whole can exist for millennia. Does this contrast also imply that the long-lived cells in a palm trunk have exceptional properties, which allows this seeming immortality? We document the long-life of many tall palm species and their inherent long-lived stem cell properties, comparing such plants to conventional trees. We provide a summary of aspects of cell age and life span in animals and plants. Cell replacement is a feature of animal function, whereas conventional trees rely on active growth centers (meristems) to sustain organismal development. However, the long persistence of living cells in palm trunks is seen not as evidence for unique metabolic processes that sustain longevity, but is a consequence of unique constructional features. This conclusion suggests that the life span of plant cells is not necessarily genetically determined.

Wollemia nobilis (Araucariaceae): Branching, vasculature and histology in juvenile stages.

We present a preliminary description of the morphology and anatomy of contrasted axes in the recently discovered conifer Wollemia nobilis, based on clonally propagated material. The novelty of this discovery and the tree's size and rarity has led to global interest and a comprehensive and successful conservation program. Our results should serve as a model on which future studies of this tree and other members of the Araucariaceae can be based. The specimens studied are mimics of the architecture of the mature tree, with precise distinction between orthotropic (= trunk) axes, which have radial symmetry (spiral phyllotaxy) and plagiotropic (= branch) axes with dorsiventral symmetry (as a modified decussate phyllotaxy). Trunk axes develop irregular pseudowhorls of branches that originate within the terminal bud by syllepsis, their extension coincident with that of the parent axis. The two kinds of axes show considerable anatomical differences, but are still based on the common feature of a single stelar-derived trace to each leaf that becomes subdivided in the cortex, a feature of the whole family. Trunk axes include extended cortical leaf and branch traces associated with abundant sclerenchyma, but branches have short cortical leaf traces, no branch traces, and limited sclerenchyma. Reiteration is limited and largely involves the formation of basal suckers on the trunk. Branches normally remain unbranched, but can do so most often when damaged. This study thus emphasizes the phenomenon of axis differentiation in conifers, which has been little investigated anatomically, but could be very important in the identification of fossils.

Evolution of lamina anatomy in the palm family (Arecaceae).

The unique properties of tree building in Arecaceae strongly constrain their architectural lability. Potentially compensating for this limitation, the extensive diversification of leaf anatomical structure within palms involves many characters whose alternate states may confer disparate mechanical or physiological capabilities. In the context of a recent global palm phylogeny, we analyzed the evolution of 10 such lamina anatomical characters and leaf morphology of 161 genera, conducting parsimony and maximum likelihood ancestral state reconstructions, as well as tests of correlated evolution. Lamina morphology evolves independently from anatomy. Although many characters do optimize as synapomorphic for major clades, anatomical evolution is highly homoplasious. Nevertheless, it is not random: analyses indicate the recurrent evolution of different cohorts of correlated character states. Notable are two surface layer (epidermis and hypodermis) types: (1) a parallel-laminated type of rectangular epidermal cells with sinuous anticlinal walls, with fibers present in the hypodermis and (2) a cross-laminated type of hexagonal cells in both layers. Correlated with the cross-laminated type is a remarkable decrease in the volume fraction of fibers, accompanied by changes in the architecture and sheath cell type of the transverse veins. We discuss these and other major patterns of anatomical evolution in relation to their biomechanical and ecophysiological significance.

Development of woody branch attachments in Schefflera (Araliaceae or Apiaceae).

Attachment of branches in Schefflera is unusual in that it involves fingerlike woody extensions that originate in the cortex and pass gradually into the woody cylinder of the parent shoot. We tested the hypothesis that these structures could be roots since Schefflera is a hemi-epiphyte with aerial roots. These branch traces originate by secondary development in the many leaf traces (LTs) of the multilacunar node together with associated accessory traces. In the primary condition, the LTs may be described as cortical bundles. Leaves are long persistent and can maintain a primary stem connection across a broad cylinder of secondary xylem. Under the stimulus of branch development, the LTs form a template for secondary vascular development. Because the LT system is broad, with many traces, the branch attachment is also broad. The fingerlike extensions are attached to the surface of the woody cylinder of the parent stem but are progressively obscured as a continuous cambium is formed. Bark tissues are included within the branch axil because of the extended cortical origin of the initial attachment. The results are discussed in the context of branch-trunk unions in tropical plants, an important component of canopy development.

Development of nonlignified fibers in leaves of Gnetum gnemon (Gnetales).

Leaves of Gnetum gnemon have an extensive anastomosing network of thick-walled cellulosic fibers that permeate mesophyll tissues. Brochidodromus venation is precise with major veins originating by uniseriate plate meristems. In mesophyll differentiation, laticifers appear before fibers and more or less parallel to major veins. Fiber initials appear later, mostly within the subhypodermal mesophyll cell layers, but otherwise adjacent to the leaf margin or the major veins. Fibers are early binucleate and sometimes become four-nucleate. Fiber initials extend by symplastic but mainly intrusive apical growth, become irregular, little branched and interpenetrate other mesophyll layers. They make frequent contact with other fibers forming the anatomosing system, but remain thin-walled until leaf expansion is complete. Sclereids are little developed, thus fibers become the main mechanical system of the mature leaf. Once expansion is complete, maturation of fibers involves rapid formation of a cellulosic but unlignified secondary wall that is non-lamellate and almost occludes the cell lumen. These fibers are contrasted with the gelatinous (tension) fibers developed eccentrically in stems of Gnetum. Apart from their mechanical function, fibers may also have a hydraulic function in maintaining a highly hydrated internal leaf atmosphere.

Development of gelatinous (reaction) fibers in stems of Gnetum gnemon (Gnetales).

In extraxylary tissues of the stem Gnetum gnemon produces gelatinous fibers that can also function as reaction or tension fibers. These gelatinous fibers occur in all axes in the outer cortex and in displaced axes progressively in the middle and inner cortex and finally in the secondary phloem. Early cell differentiation in the cortex produces initials of laticifers that are unique in gymnosperms. Subsequently narrow fibers differentiate from cells that undergo both extensive passive elongation, as a result of internodal elongation, together with their active apical intrusive growth. Outer fibers always complete secondary wall development and become an important mechanical component of stems. Differentiation of fiber initials continues in the middle and inner cortex, but secondary wall formation can only be determined by a gravimorphic stimulus that produces eccentric development of fibers. Further eccentric development of fibers then continues in the outer secondary phloem from dedifferentiated phloem parenchyma cells that initially undergo extensive intrusive growth. All such cells have characteristic features of tension fibers of angiosperms. They exhibit a pronounced purely cellulosic innermost layer of the secondary wall (Sg layer). In addition, fiber initials are coenocytic, including up to eight nuclei that become distributed uniformly throughout the length of the cell. Mature macerated fibers are markedly brittle, making accurate length measurements difficult. Although cytologically uniform, these fibers thus originate from two kinds of initial (primary and secondary). They also differ in their response to a gravimorphic stimulus determined by their times of inception and their eccentric location. These cells show a suite of positional and gravimorphic responses that illustrate the complexity of plant cell differentiation.

Tension wood fibers are related to gravitropic movement of red mangrove (Rhizophora mangle) seedlings.

Freshly collected viviparous seedlings (propagules) were collected from wild plants of Rhizophora mangle and planted in vertical or horizontal positions. A total of 80 seedlings were examined anatomically at various ages and orientations. After rooting, seedlings reoriented from horizontal to vertical by extreme bending in the hook region of the hypocotyl directly above the basal 1 cm where roots formed. Hypocotyl bending occurred over many months. Trends in position and relative abundance of tension fibers (also called gelatinous fibers) over time were followed. The erection of the seedling was related to increased secondary xylem and the number of tension wood fibers on the upper side of the hook region. However, linear regressions had low coefficient of determination (r(2)) values, presumably related to seedlings with high variability.

Developmental features of the discontinuous stem vascular system in the rattan palm Calamus (Arecaceae-Calamoideae-Calamineae).

Calamus is a climbing palm marked by considerable internodal extension and limited stem-thickening growth, but with a surprisingly discontinuous axial vascular system. Stem bundles end blindly in a basipetal direction and are connected to each other only by narrow and late-developing transverse commissures. Vascular connection via leaf traces between stem and leaf is made over about nine plastochrons (P), but the dominant central system is completed by about P(7), with subsequent bundles forming the crowded fibrous peripheral system, which has reduced or no vascular tissues. The stem internode below a leaf completes its extension and maturation only by P(10) to P(11). Axial stem bundles originate as procambial strands that are discontinuous apically for up to 15 plastochrons before being "captured" by a developing leaf. Their distal unconnected ends arise by dedifferentiation of ground parenchyma cells. Protoxylem is initiated as short overlapping initials that differentiate progressively during extension growth, which ruptures all but the last-formed elements. Their form, with tapered ends, means that they mature as tracheids. Metaxylem appears only late in shoot development, shortly before internodal elongation ceases (P(8)) and always unconnected to the late-differentiating protoxylem. In each axial bundle protophloem differentiates as a single strand, subsequently and much later appearing as two separate metaphloem strands as the early initials, ruptured by extension growth, are replaced by fibers. It is suggested that the unique features of this stem can be ascribed to the absence of a "meristematic cap," which otherwise typifies palms of normal habit, and that discontinuity is causally related to the pronounced late stem extension growth.