Developmental Genetics of Corolla Tube Formation: Role of the tasiRNA-ARF Pathway and a Conceptual Model.

Over 80,000 angiosperm species produce flowers with petals fused into a corolla tube. The corolla tube contributes to the tremendous diversity of flower morphology and plays a critical role in plant reproduction, yet it remains one of the least understood plant structures from a developmental genetics perspective. Through mutant analyses and transgenic experiments, we show that the tasiRNA-ARF pathway is required for corolla tube formation in the monkeyflower species Mimulus lewisii Loss-of-function mutations in the M. lewisii orthologs of ARGONAUTE7 and SUPPRESSOR OF GENE SILENCING3 cause a dramatic decrease in abundance of TAS3-derived small RNAs and a moderate upregulation of AUXIN RESPONSE FACTOR3 (ARF3) and ARF4, which lead to inhibition of lateral expansion of the bases of petal primordia and complete arrest of the upward growth of the interprimordial regions, resulting in unfused corollas. Using the DR5 auxin-responsive promoter, we discovered that auxin signaling is continuous along the petal primordium base and the interprimordial region during the critical stage of corolla tube formation in the wild type, similar to the spatial pattern of MlARF4 expression. Auxin response is much weaker and more restricted in the mutant. Furthermore, exogenous application of a polar auxin transport inhibitor to wild-type floral apices disrupted petal fusion. Together, these results suggest a new conceptual model highlighting the central role of auxin-directed synchronized growth of the petal primordium base and the interprimordial region in corolla tube formation.

Within-individual leaf allometry and the evolution of leaf morphology: A multilevel analysis of leaf allometry in temperate Viburnum (Adoxaceae) species.

A critical issue in evolutionary biology is understanding the relationship between macroevolutionary patterns of diversity and the origin of variation at the organismal level. Among-individual allometry, the relationship between the size and shape of a structure among organisms at a fixed developmental stage, is often similar to evolutionary allometry, the relationship between the size and shape of a structure among populations or species, and the genetic and developmental process that underlie allometric relationships at both levels are thought to influence evolutionary diversification. Metameric organisms present an additional level of allometry: the relationship between the size and shape of structures within individuals. We propose that within-individual allometry is also related to evolutionary diversification among metameric organisms. We explore this idea in temperate deciduous Viburnum (Adoxaceae) species that bear two types of leaves, that is, preformed and neoformed leaves, with contrasting patterns of development. Examination of within-individual, among-individual, among-population, and among-species allometry of leaf shape in both leaf types showed that the slopes of all allometric relationships were significantly different from isometry, and their sign was consistent across allometric hierarchies. Although the allometric slope of preformed leaves was constant across allometry levels, the allometric slope of neoformed leaves became increasingly steeper. We suggest that allometric variation underlying evolutionary diversification in metameric organisms may manifest among individuals and also among their repeated structures. Moreover, structures with contrasting patterns of development within metameric organisms can experience different degrees of developmental constraint, and this can in turn affect morphological diversification.

Preforming floral primordia converge on a narrow range of stages at dormancy despite multiple effects of temperature on development.

Phenological studies often focus on relationships between flowering date and temperature or other environmental variables. Yet in species that preform flowers, anthesis is one stage of a lengthy developmental process, and effects of temperature on flower development in the year(s) before flowering are largely unknown. We investigated the effects of temperature during preformation on flower development in Vaccinium vitis-idaea. Using scanning electron microscopy, we established scores for developing primordia and examined effects of air temperature, depth of soil thaw, time of year and previous stage on development. Onset of flower initiation depends on soil thaw, and developmental change is greatest at early stages and during the warmest months. Regardless of temperature and time during the season, all basal floral primordia pause development at the same stage before whole-plant dormancy. Once primordia are initiated, development does not appear to be influenced by air temperature differences within the range of variation among our sites. There may be strong endogenous flower-level controls over development, particularly the stage at which morphogenesis ceases before dormancy. However, the strength of such internal controls in the face of continuing temperature extremes under a changing climate is unclear.

Comparative analysis of corolla tube development across three closely related Mimulus species with different pollination syndromes.

Fusion of petals to form a corolla tube is considered a key innovation contributing to the diversification of many flowering plant lineages. Corolla tube length often varies dramatically among species and is a major determinant of pollinator preference. However, our understanding of the developmental dynamics underlying corolla tube length variation is very limited. Here we examined corolla tube growth in the Mimulus lewisii species complex, an emerging model system for studying the developmental genetics and evo-devo of pollinator-associated floral traits. We compared developmental and cellular processes associated with corolla tube length variation among the bee-pollinated M. lewisii, the hummingbird-pollinated Mimulus verbenaceus, and the self-pollinated Mimulus parishii. We found that in all three species, cell size is non-uniformly distributed along the mature tube, with the longest cells just distal to the stamen insertion site. Differences in corolla tube length among the three species are not associated with processes of organogenesis or early development but are associated with variation in multiple processes occurring later in development, including the location and duration of cell division and cell elongation. The tube growth curves of the small-flowered M. parishii and large-flowered M. lewisii are essentially indistinguishable, except that M. parishii tubes stop growing earlier at a smaller size, suggesting a critical role of heterochrony in the shift from outcrossing to selfing. These results not only highlight the developmental process associated with corolla tube variation among species but also provide a baseline reference for future developmental genetic analyses of mutants or transgenic plants with altered corolla tube morphology in this emerging model system.

Evidence for parent-of-origin effects and interparental conflict in seeds of an ancient flowering plant lineage.

Theoretical and empirical studies have long connected the evolutionary innovation of endosperm, a genetically biparental product of a double fertilization process unique to flowering plants (angiosperms), to conflicting parental interests over offspring provisioning. Yet, none of these studies examined interparental conflict in representatives of any of the most ancient angiosperm lineages. We performed reciprocal interploidy crosses in the water lily Nymphaea thermarum, a member of one of the most ancient angiosperm lineages, Nymphaeales. We find that an excess of paternal genomes is associated with an increase in endosperm growth. By contrast, maternal ploidy negatively influences development or growth of all seed components, regardless of paternal genome dosage. Most relevant to the conflict over distribution of maternal resources, however, is that growth of the perisperm (seed storage tissue derived from the maternal sporophyte, found in all Nymphaeales) is unaffected by paternal genome dosage-ensuring maternal control of maternal resources. We conclude that the evolutionary transfer of embryo-nourishing function from a genetically biparental endosperm to a genetically maternal perisperm can be viewed as an effective maternal strategy to recapture control of resource distribution among progeny, and thus that interparental conflict has influenced the evolution of seed development in this ancient angiosperm lineage.

Contrasting lengths of Pelargonium floral nectar tubes result from late differences in rate and duration of growth.

Background and Aims: Much of morphological evolution in flowers has arisen from pollinator-mediated selection, often manifest as a match between the length of the pollinator's proboscis and the depth of tubular corollas or spurs. We investigate development, growth and homology of the unique nectar tube of Pelargonium, frequently described as 'a spur adnate to the pedicel'. Methods: We focused on two species. The nectar tube of P. ionidiflorum is three times longer than that of P. odoratissimum. Light and scanning electron microscopy were carried out, and daily growth measurements were used to compare nectar tube development and vascular patterns. Key Results: Nectar tubes in both species are initiated centripetally to the dorsal sepal in a space created by lateral displacement of two antepetalous stamens. The cavity deepens through subsequent intercalary growth of the receptacle that proceeds at the same rate in both species until tubes reach approx. 10 mm in length. Differences in final nectar tube lengths arise via an increase in the rate and duration of growth of the receptacle that begins just before anthesis (floral opening) and continues for several days past anthesis in P. ionidiflorum but does not occur in P. odoratissimum. Epidermal cells of the dorsal surface of the nectar tube in P. ionidiflorum are approx. 1.6 times longer than those in P. odoratissimum. Histological sections show no evidence that the nectar tube is a spur that became evolutionarily fused to the pedicel. Conclusions: Nectar tubes in Pelargonium are localized cavities that form in the receptacle via intercalary growth. Differences in the rate and duration of growth just prior to and following anthesis underlie differences in final tube lengths. Because differences in cell lengths do not fully account for differences in nectar tube lengths, evolutionary diversification must involve changes in both cell cycle and cell expansion.

Non-equilibrium dynamics and floral trait interactions shape extant angiosperm diversity.

Why are some traits and trait combinations exceptionally common across the tree of life, whereas others are vanishingly rare? The distribution of trait diversity across a clade at any time depends on the ancestral state of the clade, the rate at which new phenotypes evolve, the differences in speciation and extinction rates across lineages, and whether an equilibrium has been reached. Here we examine the role of transition rates, differential diversification (speciation minus extinction) and non-equilibrium dynamics on the evolutionary history of angiosperms, a clade well known for the abundance of some trait combinations and the rarity of others. Our analysis reveals that three character states (corolla present, bilateral symmetry, reduced stamen number) act synergistically as a key innovation, doubling diversification rates for lineages in which this combination occurs. However, this combination is currently less common than predicted at equilibrium because the individual characters evolve infrequently. Simulations suggest that angiosperms will remain far from the equilibrium frequencies of character states well into the future. Such non-equilibrium dynamics may be common when major innovations evolve rarely, allowing lineages with ancestral forms to persist, and even outnumber those with diversification-enhancing states, for tens of millions of years.

Kin recognition within a seed and the effect of genetic relatedness of an endosperm to its compatriot embryo on maize seed development.

As one of two sexual products resulting from double fertilization in angiosperms, the endosperm nourishes its compatriot embryo during seed development and/or germination and ultimately dies. Theoretical studies suggest that the genetic relatedness of an endosperm to its embryo in the same seed might determine the amount of resources ultimately available for the embryo during seed development. We took advantage of the phenomenon of heterofertilization in cultivated maize to empirically test whether genetic relatedness between a triploid embryo-nourishing endosperm and its compatriot diploid embryo impacts the process of resource allocation between these two sexually produced entities. We used genetically distinct maize inbred lines to perform two crossing experiments. Dry weights of dissected embryos and endosperms of mature heterofertilized and adjacent homofertilized kernels were compared. Embryo weight of heterofertilized kernels was significantly less than that of embryos of homofertilized kernels, whereas there was no significant difference in endosperm weight between the two types of kernels. Our results suggest that the degree of genetic relatedness of an endosperm to its compatriot embryo affects seed development and specifically the amount of maternal resources allocated to an endosperm that are eventually turned over to an embryo. The lower the coefficient of relatedness of an endosperm to its compatriot embryo, the smaller the embryo. Thus, the endosperm of a heterofertilized seed appears to behave less cooperatively with respect to resource transfer toward its less closely related embryo compared with the endosperm of a homofertilized seed.

Multiple developmental processes underlie sex differentiation in angiosperms.

The production of unisexual flowers has evolved numerous times in dioecious and monoecious plant taxa. Based on repeated evolutionary origins, a great variety of developmental and genetic mechanisms underlying unisexual flower development is predicted. Here, we comprehensively review the modes of development of unisexual flowers, test potential correlations with sexual system, and end with a synthesis of the genetics and hormonal regulation of plant sex determination. We find that the stage of organ abortion in male and female flowers is temporally correlated within species and also confirm that the arrest of development does not tend to occur preferentially at a particular stage, or via a common process.

Charles Darwin and the origins of plant evolutionary developmental biology.

Much has been written of the early history of comparative embryology and its influence on the emergence of an evolutionary developmental perspective. However, this literature, which dates back nearly a century, has been focused on metazoans, without acknowledgment of the contributions of comparative plant morphologists to the creation of a developmental view of biodiversity. We trace the origin of comparative plant developmental morphology from its inception in the eighteenth century works of Wolff and Goethe, through the mid nineteenth century discoveries of the general principles of leaf and floral organ morphogenesis. Much like the stimulus that von Baer provided as a nonevolutionary comparative embryologist to the creation of an evolutionary developmental view of animals, the comparative developmental studies of plant morphologists were the basis for the first articulation of the concept that plant (namely floral) evolution results from successive modifications of ontogeny. Perhaps most surprisingly, we show that the first person to carefully read and internalize the remarkable advances in the understanding of plant morphogenesis in the 1840s and 1850s is none other than Charles Darwin, whose notebooks, correspondence, and (then) unpublished manuscripts clearly demonstrate that he had discovered the developmental basis for the evolutionary transformation of plant form.

Developmental plasticity, genetic assimilation, and the evolutionary diversification of sexual expression in Solanum.

PREMISE OF THE STUDY: For over a century, it has been hypothesized that selection can convert an environmentally induced phenotype (i.e., plasticity) into a fixed (constitutively produced) phenotype, a process known as genetic assimilation. While evidence of assimilation is accumulating, the role of plasticity generally and assimilation specifically in evolutionary diversification has rarely been examined from a comparative phylogenetic perspective. • METHODS: We combined experimental analyses of plasticity with ancestral state reconstructions to examine the evolutionary dynamics of sexual expression in two well-characterized sections (Acanthophora and Lasiocarpa) in Solanum subgenus Leptostemonum. We examined sexual expression phenotypes and the proportion of staminate flowers produced under contrasting resource conditions in 10 species and combined these data with previous studies. • KEY RESULTS: Staminate flower production was phenotypically plastic for nine of 14 species and unaffected by treatment in five species. Two of the nonplastic species bore few staminate flowers, and three constitutively produced large numbers of staminate flowers. For individuals and species producing staminate flowers, these flowers occurred in a distinctive architectural pattern that was qualitatively the same in both plastic and nonplastic species. Parsimony and Bayesian reconstructions demonstrate that plasticity is ancestral among the species studied. • CONCLUSIONS: Plasticity has been lost independently in sections Acanthophora and Lasiocarpa, and the consequence of its loss results in evolutionary diversification of sexual expression. In section Acanthophora, loss of plasticity represents a reversion to production of predominantly hermaphroditic flowers. In contrast, the fixed production of staminate flowers in Lasiocarpa has the hallmarks of evolution via genetic assimilation.

Flower heterochrony and crop yield.

Crop improvement has focused on enhancing yield, nutrient content, harvestability, and stress resistance using a trait-centered reductionist approach. This has downplayed the fact that plants are developmentally integrated and respond coordinately and predictably to genetic and environmental variation, with potential consequences for food production. Crop yield, including both fruit/seed production and the possibility of generating hybrid crop varieties, is highly dependent on flower morphology and sex, which, in turn, can be profoundly affected by slight shifts in the timing and rate of flower organ development (i.e., flower heterochrony). We argue that understanding the genetic and environmental bases of flower heterochrony and their effect on flower morphology and sex in cultivated plants and in their wild relatives can facilitate crop improvement.

Patterns of shoot architecture in locally adapted populations are linked to intraspecific differences in gene regulation.

• Shoot architecture, including the number and location of branches, is a crucial aspect of plant function, morphological diversification, life history evolution and crop domestication. • Genes controlling shoot architecture are well characterized in, and largely conserved across, model flowering plant species. The role of these genes in the evolution of morphological diversity in natural populations, however, has not been explored. • We identify axillary meristem outgrowth as a primary driver of divergent branch number and life histories in two locally adapted populations of the monkeyflower, Mimulus guttatus. • Furthermore, we show that MORE AXILLARY GROWTH (MAX) gene expression strongly correlates with natural variation in branch outgrowth in this species, linking modification of the MAX-dependent pathway to the evolutionary diversification of shoot architecture.

Modular regulation of floral traits by a PRE1 homolog in Mimulus verbenaceus: implications for the role of pleiotropy in floral integration.

Floral traits often show correlated variation within and among species. For species with fused petals, strong correlations among corolla tube, stamen, and pistil length are particularly prevalent, and these three traits are considered an intra-floral functional module. Pleiotropy has long been implicated in such modular integration of floral traits, but empirical evidence based on actual gene function is scarce. We tested the role of pleiotropy in the expression of intra-floral modularity in the monkeyflower species Mimulus verbenaceus by transgenic manipulation of a homolog of Arabidopsis PRE1. Downregulation of MvPRE1 by RNA interference resulted in simultaneous decreases in the lengths of corolla tube, petal lobe, stamen, and pistil, but little change in calyx and leaf lengths or organ width. Overexpression of MvPRE1 caused increased corolla tube and stamen lengths, with little effect on other floral traits. Our results suggest that genes like MvPRE1 can indeed regulate multiple floral traits in a functional module but meanwhile have little effect on other modules, and that pleiotropic effects of these genes may have played an important role in the evolution of floral integration and intra-floral modularity.

Female gametophyte development and double fertilization in Balsas teosinte, Zea mays subsp. parviglumis (Poaceae).

Over the course of maize evolution, domestication played a major role in the structural transition of the vegetative and reproductive characteristics that distinguish it from its closest wild relative, Zea mays subsp. parviglumis (Balsas teosinte). Little is known, however, about impacts of the domestication process on the cellular features of the female gametophyte and the subsequent reproductive events after fertilization, even though they are essential components of plant sexual reproduction. In this study, we investigated the developmental and cellular features of the Balsas teosinte female gametophyte and early developing seed in order to unravel the key structural and evolutionary transitions of the reproductive process associated with the domestication of the ancestor of maize. Our results show that the female gametophyte of Balsas teosinte is a variation of the Polygonum type with proliferative antipodal cells and is similar to that of maize. The fertilization process of Balsas teosinte also is basically similar to domesticated maize. In contrast to maize, many events associated with the development of the embryo and endosperm appear to be initiated earlier in Balsas teosinte. Our study suggests that the pattern of female gametophyte development with antipodal proliferation is common among species and subspecies of Zea and evolved before maize domestication. In addition, we propose that the relatively longer duration of the free nuclear endosperm phase in maize is correlated with the development of a larger fruit (kernel or caryopsis) and with a bigger endosperm compared with Balsas teosinte.

Node-specific branching and heterochronic changes underlie population-level differences in Mimulus guttatus (Phrymaceae) shoot architecture.

PREMISE OF THE STUDY: Shoot architecture is a fundamentally developmental aspect of plant biology with implications for plant form, function, reproduction, and life history evolution. Mimulus guttatus is morphologically diverse and becoming a model for evolutionary biology. Shoot architecture, however, has never been studied from a developmental perspective in M. guttatus. METHODS: We examined the development of branches and flowers in plants from two locally adapted populations of M. guttatus with contrasting flowering times, life histories, and branch numbers. We planted second-generation seed in growth chambers to control for maternal and environmental effects. KEY RESULTS: Most branches occurred at nodes one and two of the main axis. Onset of branching occurred earlier and at a greater frequency in perennials than in annuals. In perennials, almost all flowers occurred at the fifth or more distal nodes. In annuals, most flowers occurred at the third and more distal nodes. Accessory axillary meristems and higher-order branching did not influence shoot architecture. CONCLUSIONS: We found no evidence for trade-offs between flowers and branches because axillary meristem number was not limiting: a large number of meristems remained quiescent. If, however, quiescence is a component of meristem allocation strategy, then meristems may be limited despite presence of quiescent meristems. At the two basalmost nodes, branch number was determined by mechanisms governing either meristem initiation or outgrowth, rather than flowering vs. branching. At the third and more distal nodes, heterochronic processes contributed to flowering time and branch number differences between populations.