Compound leaf development in the palm Chamaedorea elegans is KNOX-independent.

PREMISE OF THE STUDY: How a leaf acquires its shape is a major and largely unresolved question in plant biology. This problem is particularly complex in the case of compound leaves, where the leaf blade is subdivided into leaflets. In many eudicots with compound leaves, class I KNOTTED1-LIKE HOMEOBOX (KNOX) genes are upregulated in the leaf primordium and promote leaflet initiation, while KNOX genes are restricted to the shoot apical meristem in simple-leaved plants. In monocots, however, little is known about the extent of KNOX contribution to compound leaf development, and we aimed to address this issue in the palm Chamaedorea elegans. METHODS: We investigated the accumulation pattern of KNOX proteins in shoot apical meristems and leaf primordia of the palm C. elegans using immunolocalization experiments. KEY RESULTS: KNOX proteins accumulated in vegetative and inflorescence apical meristems and in the subtending stem tissue, but not in the plicated regions of the leaf primordia. These plicated areas form during primary morphogenesis and are the only meristematic tissue in the developing primordium. In addition, KNOX proteins did not accumulate in any region of the developing leaf during secondary morphogenesis, when leaflets separate to create the final pinnately compound leaf. CONCLUSIONS: The compound leaf character in palms, C. elegans in particular and likely other pinnately compound palms, does not depend on the activities of KNOX proteins.

Characterization of C3--C4 intermediate species in the genus Heliotropium L. (Boraginaceae): anatomy, ultrastructure and enzyme activity.

Photosynthetic pathway characteristics were studied in nine species of Heliotropium (sensu lato, including Euploca), using assessments of leaf anatomy and ultrastructure, activities of PEP carboxylase and C4 acid decarboxylases, and immunolocalization of ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) and the P-subunit of glycine decarboxylase (GDC). Heliotropium europaeum, Heliotropium calcicola and Heliotropium tenellum are C3 plants, while Heliotropium texanum and Heliotropium polyphyllum are C4 species. Heliotropium procumbens and Heliotropium karwinskyi are functionally C3, but exhibit 'proto-Kranz' anatomy where bundle sheath (BS) cells are enlarged and mitochondria primarily occur along the centripetal (inner) wall of the BS cells; GDC is present throughout the leaf. Heliotropium convolvulaceum and Heliotropium greggii are C3--C4 intermediates, with Kranz-like enlargement of the BS cells, localization of mitochondria along the inner BS wall and a loss of GDC in the mesophyll (M) tissue. These C3--C4 species of Heliotropium probably shuttle photorespiratory glycine from the M to the BS tissue for decarboxylation. Heliotropium represents an important new model for studying C4 evolution. Where existing models such as Flaveria emphasize diversification of C3--C4 intermediates, Heliotropium has numerous C3 species expressing proto-Kranz traits that could represent a critical initial phase in the evolutionary origin of C4 photosynthesis.

Shifts in leaf vein density through accelerated vein formation in C4 Flaveria (Asteraceae).

BACKGROUND AND AIMS: Leaf venation in many C(4) species is characterized by high vein density, essential in facilitating rapid intercellular diffusion of C(4) photosynthetic metabolites between different tissues (mesophyll, bundle sheath). Greater vein density has been hypothesized to be an early step in C(4) photosynthesis evolution. Development of C(4) vein patterning is thought to occur from either accelerated or prolonged procambium formation, relative to ground tissue development. METHODS: Cleared and sectioned tissues of phylogenetically basal C(3) Flaveria robusta and more derived C(4) Flaveria bidentis were compared for vein pattern in mature leaves and vein pattern formation in developing leaves. KEY RESULTS: In mature leaves, major vein density did not differ between C(3) and C(4) Flaveria species, whereas minor veins were denser in C(4) species than in C(3) species. The developmental study showed that both major and minor vein patterning in leaves of C(3) and C(4) species were initiated at comparable stages (based on leaf length). An additional vein order in the C(4) species was observed during initiation of the higher order minor veins compared with the C(3) species. In the two species, expansion of bundle sheath and mesophyll cells occurred after vein pattern was complete and xylem differentiation was continuous in minor veins. In addition, mesophyll cells ceased dividing sooner and enlarged less in C(4) species than in C(3) species. CONCLUSIONS: Leaf vein pattern characteristic to C(4) Flaveria was achieved primarily through accelerated and earlier offset of higher order vein formation, rather than other modifications in the timing of vein pattern formation, as compared with C(3) species. Earlier cessation of mesophyll cell division and reduced expansion also contributed to greater vein density in the C(4) species. The relatively late expansion of bundle sheath and mesophyll cells shows that vein patterning precedes ground tissue development in C(4) species.

Modification of cell proliferation patterns alters leaf vein architecture in Arabidopsis thaliana.

Formation of leaf vascular pattern requires regulation of a number of cellular processes, including cell proliferation. To assess the role of cell proliferation during vein order formation, leaf development in genetic lines exhibiting aberrant cell proliferation patterns due to altered expression patterns of ANT and ICK1 genes was analyzed. Modification of cell proliferation patterns alters the number of higher order veins and the number of minor tertiary veins remodeled as intersecondary veins in Arabidopsis rosette leaves. Minor vein complexity, as indicated by branch point and freely ending veinlet number, is highly correlated with a decrease or increase in cell proliferation. Observations of procambial strand formation in modified cell proliferation pattern lines showed that vein pattern is specified early in leaf development and that formation of freely ending veinlets is temporally correlated with the expansion of ground meristem when cell proliferation is terminated prematurely. Taken together, our observations indicate that: (1) genes that modulate cell proliferation play a key role in regulating the meristematic competence of ground meristem cells to form procambium and vein pattern during leaf development, and (2) ANT is a crucial part of this regulation.

Diversity of Kranz anatomy and biochemistry in C4 eudicots.

C(4) photosynthesis and Kranz anatomy occur in 16 eudicot families, a striking example of convergent evolution. Biochemical subtyping for 13 previously undiagnosed C(4) eudicot species indicated that 10 were NADP-malic enzyme (ME) and three were NAD-ME. A total of 33 C(4) species, encompassing four Kranz anatomical types (atriplicoid, kochioid, salsoloid, and suaedioid), and 21 closely related C(3) species were included in a quantitative anatomical study in which we found that, unlike similar studies in grasses and sedges, anatomical type had no predictive value for the biochemical subtype. In a multivariate canonical discriminant analysis, C(4) species were distinguished from C(3) species by the mesophyll to bundle sheath ratio and exposure of the bundle sheath surface to intercellular space. Discrimination between NADP-ME and NAD-ME was not significant, although in a Mantel test grouping by biochemical subtype was significant, while grouping by family was not. This comprehensive survey of C(4) anatomy and biochemistry unequivocally demonstrated that atriplicoid anatomy and NADP-ME biochemistry predominate in many evolutionary lineages. In addition to a main decarboxylating enzyme, high activity of a second decarboxylating enzyme was often observed. Notably, PEP-carboxykinase activity was significant in a number of species, demonstrating that this enzyme could also serve as a secondary pathway for C(4) metabolism in eudicots.

Key innovations in the evolution of Kranz anatomy and C4 vein pattern in Flaveria (Asteraceae).

Kranz anatomy and C(4) vein pattern are required for C(4) biochemical functioning in C(4) plants; however, the evolutionary timing of anatomical and biochemical adaptations is unknown. From the genus Flaveria, 16 species (C(3), C(4), intermediates [C(3)-C(4), C(4)-like]) were analyzed, novel anatomical and vein pattern characters were analyzed and key anatomical differences among photosynthetic groups were highlighted. A stepwise acquisition of anatomical and vein pattern traits prior to derived biochemistry was outlined on the basis of the phylogeny of Flaveria. Increased vein density represents a potential "precondition" contributing to lower ratios of photosynthetic tissues (mesophyll, bundle sheath) and precedes further anatomical and biochemical modifications observed in derived C(3)-C(4) intermediates. In derived Flaveria species, bundle sheath volume is modified through cell expansion, whereas mesophyll volume is altered through mesophyll cell expansion, reductions in the number of ground tissue layers, and increased vein density. Results demonstrated that key anatomical features of C(4) plants are also required for C(3)-C(4) biochemical intermediacy, and anatomical and biochemical alterations acquired during evolution of intermediacy may predispose a species for evolution of C(4) photosynthesis. C(4)-like species are similar to C(4) species, demonstrating that Kranz anatomy is fully evolved before complete C(4) biochemistry is achieved.

Cell wall degradation and modification during programmed cell death in lace plant, Aponogeton madagascariensis (Aponogetonaceae).

An unusual form of leaf morphogenesis occurs in the aquatic, lace plant, Aponogeton madagascariensis (Aponogetonaceae). Early in development, discrete patches of cells undergo programmed cell death (PCD) and form perforations during leaf expansion. In addition to the protoplasts, walls of the dying cells are degraded during PCD. The cuticle of the perforation site is eroded first, followed by dissolution of cell wall matrix components, so that walls appear as loose fibrillar networks as perforations form. Gel diffusion assays of wall-degrading enzyme activity indicated that pectinases are active throughout leaf development, while cellulase activity was restricted to early stages of perforation formation. Alcian blue staining showed that degrading walls remain rich in pectin, and immunolocalization of pectin epitopes indicated that the proportions of esterified and de-esterifed pectins do not change significantly. Walls of perforation border cells are modified by suberin deposition late in development, and reactive oxygen species, thought to have a role in polymerization of phenolic suberin monomers, are present at the same stage. This timing suggests that suberization may limit the spread of PCD and provide an apoplastic barrier against microbial invasion but does not initiate PCD.

Programmed cell death and leaf morphogenesis in Monstera obliqua (Araceae).

The unusual perforations in the leaf blades of Monstera obliqua (Araceae) arise through programmed cell death early in leaf development. At each perforation site, a discrete subpopulation of cells undergoes programmed cell death simultaneously, while neighboring protoderm and ground meristem cells are unaffected. Nuclei of cells within the perforation site become terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive, indicating that DNA cleavage is an early event. Gel electrophoresis indicates that DNA cleavage is random and does not result in bands that represent multiples of internucleosomal units. Ultrastructural analysis of cells at the same stage reveals misshapen, densely stained nuclei with condensed chromatin, disrupted vacuoles, and condensed cytoplasm. Cell walls within the perforation site remain intact, although a small disk of dying tissue becomes detached from neighboring healthy tissues as the leaf expands and stretches the minute perforation. Exposed ground meristem cells at the rim of the perforation differentiate as epidermal cells. The cell biology of perforation formation in Monstera resembles that in the aquatic plant Aponogeton madagascariensis (Aponogetonaceae; Gunawardena et al. 2004), but the absence of cell wall degradation and the simultaneous execution of programmed cell death throughout the perforation site reflect the convergent evolution of this distinct mode of leaf morphogenesis in these distantly related plants.

Phylogeny of Flaveria (Asteraceae) and inference of C4 photosynthesis evolution.

A well-resolved phylogeny of Flaveria is used to infer evolutionary relationships among species, biogeographical distributions, and C(4) photosynthetic evolution. Data on morphology, life history, and DNA sequences (chloroplastic trnL-F, nuclear ITS and ETS) for 21 of 23 known species were collected. Each data set was analyzed separately and in combination using maximum parsimony and Bayesian analyses. The phylogeny of Flaveria is based on the combined analysis of all data. Our phylogenetic evidence indicates that C(3) Flaveria are all basal to intermediate (C(3)-C(4) and C(4)-like) and fully expressed C(4) Flaveria species. Two strongly supported clades (A and B) are present. Using this phylogeny, we evaluate the current systematics of the genus and suggest the removal and reevaluation of certain taxa. We also infer the center of origin and dispersal of Flaveria species. Multiple origins of photosynthetic pathway intermediacy in Flaveria are recognized. C(3)-C(4) intermediacy has evolved twice in the genus and is found to be evolutionarily intermediate in clade A, but not necessarily in clade B. C(4)-like photosynthesis is also derived once in each clade. In addition, fully expressed C(4) photosynthesis may have evolved up to three times within clade A.

Programmed cell death remodels lace plant leaf shape during development.

Programmed cell death (PCD) functions in the developmental remodeling of leaf shape in higher plants, a process analogous to digit formation in the vertebrate limb. In this study, we provide a cytological characterization of the time course of events as PCD remodels young expanding leaves of the lace plant. Tonoplast rupture is the first PCD event in this system, indicated by alterations in cytoplasmic streaming, loss of anthocyanin color, and ultrastructural appearance. Nuclei become terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling positive soon afterward but do not become morphologically altered until late stages of PCD. Genomic DNA is fragmented, but not into internucleosomal units. Other cytoplasmic changes, such as shrinkage and degradation of organelles, occur later. This form of PCD resembles tracheary element differentiation in cytological execution but requires unique developmental regulation so that discrete panels of tissue located equidistantly between veins undergo PCD while surrounding cells do not.

The development of enantiostyly.

Enantiostyly, the deflection of the style either to the left (left-styled) or right (right-styled) side of the floral axis, has evolved in at least ten angiosperm families. Two types of enantiostyly occur: monomorphic enantiostyly, in which individuals exhibit both stylar orientations, and dimorphic enantiostyly, in which the two stylar orientations occur on separate plants. To evaluate architectural or developmental constraints on the evolution of both forms of enantiostyly, we examined inflorescence structure and floral development among unrelated enantiostylous species. We investigated relations between the position of left- and right-styled flowers and inflorescence architecture in four monomorphic enantiostylous species, and we examined the development of enantiostyly in nine monomorphic and dimorphic enantiostylous species from five unrelated lineages. The location of left- and right-styled flowers within inflorescences ranged from highly predictable (in Solanum rostratum) to random (in Heteranthera mexicana). There were striking differences among taxa in the timing of stylar bending. In Wachendorfia paniculata, Dilatris corymbosa, and Philydrum lanuginosum, the style deflected in the bud, whereas in Heteranthera spp., Monochoria australasica, Cyanella lutea, and Solanum rostratum, stylar bending occurred at the beginning of anthesis. Comparisons of organ initiation and development indicated that asymmetries along the left-right axis were expressed very late in development, despite the early initiation of a dorsiventral asymmetry. We suggest that the evolution of dimorphic enantiostyly from monomorphic enantiostyly may be constrained by a lack of left-right positional information in the bud.