PREMISE:Phylogenetic trees of bryophytes provide important evolutionary context for land plants. However, published inferences of overall embryophyte relationships vary considerably. We performed phylogenomic analyses of bryophytes and relatives using both mitochondrial and plastid gene sets, and investigated bryophyte plastome evolution.METHODS:We employed diverse likelihood-based analyses to infer large-scale bryophyte phylogeny for mitochondrial and plastid data sets. We tested for changes in purifying selection in plastid genes of a mycoheterotrophic liverwort (Aneura mirabilis) and a putatively mycoheterotrophic moss (Buxbaumia), and compared 15 bryophyte plastomes for major structural rearrangements.RESULTS:Overall land-plant relationships conflict across analyses, generally weakly. However, an underlying (unrooted) four-taxon tree is consistent across most analyses and published studies. Despite gene coverage patchiness, relationships within mosses, liverworts, and hornworts are largely congruent with previous studies, with plastid results generally better supported. Exclusion of RNA edit sites restores cases of unexpected non-monophyly to monophyly for Takakia and two hornwort genera. Relaxed purifying selection affects multiple plastid genes in mycoheterotrophic Aneura but not Buxbaumia. Plastid genome structure is nearly invariant across bryophytes, but the tufA locus, presumed lost in embryophytes, is unexpectedly retained in several mosses.CONCLUSIONS:A common unrooted tree underlies embryophyte phylogeny, [(liverworts, mosses), (hornworts, vascular plants)]; rooting inconsistency across studies likely reflects substantial distance to algal outgroups. Analyses combining genomic and transcriptomic data may be misled locally for heavily RNA-edited taxa. The Buxbaumia plastome lacks hallmarks of relaxed selection found in mycoheterotrophic Aneura. Autotrophic bryophyte plastomes, including Buxbaumia, hardly vary in overall structure.

Land plant bodies develop from meristems, groups of pluripotent stem cells, which may persist throughout the life of a plant or, alternatively, have a transitory existence. Early diverging land plants exhibit indeterminate (persistent) growth in their haploid gametophytic generation, whereas later diverging lineages exhibit indeterminate growth in their diploid sporophytic generation, raising the question of whether genetic machinery directing meristematic functions was co-opted between generations. Class III HD-Zip (C3HDZ) genes are required for the establishment and maintenance of shoot apical meristems in flowering plants. We demonstrate that in the moss Physcomitrella patens, C3HDZ genes are expressed in transitory meristems in both the gametophytic and sporophytic generations, but not in the persistent shoot meristem of the gametyphyte. Loss-of-function of P. patens C3HDZ was engineered using ectopic expression of miR166, an endogenous regulator of C3HDZ gene activity. Loss of C3HDZ gene function impaired the function of gametophytic transitory meristematic activity but did not compromise the functioning of the persistent shoot apical meristem during the gametophyte generation. These results argue against a wholesale co-option of meristematic gene regulatory networks from the gametophyte to the sporophyte during land plant evolution, instead suggesting that persistent meristems with a single apical cell in P. patens and persistent complex meristems in flowering plants are regulated by different genetic programs.

Members of the YABBY gene family of transcription factors in angiosperms have been shown to be involved in the initiation of outgrowth of the lamina, the maintenance of polarity, and establishment of the leaf margin. Although most of the dorsal-ventral polarity genes in seed plants have homologs in non-spermatophyte lineages, the presence of YABBY genes is restricted to seed plants. To gain insight into the origin and diversification of this gene family, we reconstructed the evolutionary history of YABBY gene lineages in seed plants. Our findings suggest that either one or two YABBY genes were present in the last common ancestor of extant seed plants. We also examined the expression of YABBY genes in the gymnosperms Ephedra distachya (Gnetales), Ginkgo biloba (Ginkgoales), and Pseudotsuga menziesii (Coniferales). Our data indicate that some YABBY genes are expressed in a polar (abaxial) manner in leaves and female cones in gymnosperms. We propose that YABBY genes already acted as polarity genes in the last common ancestor of extant seed plants.

It is commonly believed that gene duplications provide the raw material for morphological evolution. Both the number of genes and size of gene families have increased during the diversification of land plants. Several small proteins that regulate transcription factors have recently been identified in plants, including the LITTLE ZIPPER (ZPR) proteins. ZPRs are post-translational negative regulators, via heterodimerization, of class III Homeodomain Leucine Zipper (C3HDZ) proteins that play a key role in directing plant form and growth. We show that ZPR genes originated as a duplication of a C3HDZ transcription factor paralog in the common ancestor of euphyllophytes (ferns and seed plants). The ZPRs evolved by degenerative mutations resulting in loss all of the C3HDZ functional domains, except the leucine zipper that modulates dimerization. ZPRs represent a novel regulatory module of the C3HDZ network unique to the euphyllophyte lineage, and their origin correlates to a period of rapid morphological changes and increased complexity in land plants. The origin of the ZPRs illustrates the significance of gene duplications in creating developmental complexity during land plant evolution that likely led to morphological evolution.

Class IV homeodomain leucine zipper (C4HDZ) genes are plant-specific transcription factors that, based on phenotypes in Arabidopsis thaliana, play an important role in epidermal development. In this study, we sampled all major extant lineages and their closest algal relatives for C4HDZ homologs and phylogenetic analyses result in a gene tree that mirrors land plant evolution with evidence for gene duplications in many lineages, but minimal evidence for gene losses. Our analysis suggests an ancestral C4HDZ gene originated in an algal ancestor of land plants and a single ancestral gene was present in the last common ancestor of land plants. Independent gene duplications are evident within several lineages including mosses, lycophytes, euphyllophytes, seed plants, and, most notably, angiosperms. In recently evolved angiosperm paralogs, we find evidence of pseudogenization via mutations in both coding and regulatory sequences. The increasing complexity of the C4HDZ gene family through the diversification of land plants correlates to increasing complexity in epidermal characters.

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We report the genome sequence of the nonseed vascular plant, Selaginella moellendorffii, and by comparative genomics identify genes that likely played important roles in the early evolution of vascular plants and their subsequent evolution

Vascular plants appeared ~410 million years ago, then diverged into several lineages of which only two survive: the euphyllophytes (ferns and seed plants) and the lycophytes. We report here the genome sequence of the lycophyte Selaginella moellendorffii (Selaginella), the first nonseed vascular plant genome reported. By comparing gene content in evolutionarily diverse taxa, we found that the transition from a gametophyte- to a sporophyte-dominated life cycle required far fewer new genes than the transition from a nonseed vascular to a flowering plant, whereas secondary metabolic genes expanded extensively and in parallel in the lycophyte and angiosperm lineages. Selaginella differs in posttranscriptional gene regulation, including small RNA regulation of repetitive elements, an absence of the trans-acting small interfering RNA pathway, and extensive RNA editing of organellar genes.

In seed plants, leaves are born on radial shoots, but unlike shoots, they are determinate dorsiventral organs made of flat lamina. YABBY genes are found only in seed plants and in all cases studied are expressed primarily in lateral organs and in a polar manner. Despite their simple expression, Arabidopsis thaliana plants lacking all YABBY gene activities have a wide range of morphological defects in all lateral organs as well as the shoot apical meristem (SAM). Here, we show that leaves lacking all YABBY activities are initiated as dorsiventral appendages but fail to properly activate lamina programs. In particular, the activation of most CINCINNATA-class TCP genes does not commence, SAM-specific programs are reactivated, and a marginal leaf domain is not established. Altered distribution of auxin signaling and the auxin efflux carrier PIN1, highly reduced venation, initiation of multiple cotyledons, and gradual loss of the SAM accompany these defects. We suggest that YABBY functions were recruited to mold modified shoot systems into flat plant appendages by translating organ polarity into lamina-specific programs that include marginal auxin flow and activation of a maturation schedule directing determinate growth.

The fossil record reveals that seed plant leaves evolved from ancestral lateral branch systems. Over time, the lateral branch systems evolved to become determinate, planar and eventually laminar. Considering their evolutionary histories, it is instructive to compare the developmental genetics of shoot apical meristems (SAMs) and leaves in extant seed plants. Genetic experiments in model angiosperm species have assigned functions of meristem maintenance, specification of stem cell identity, boundary formation, polarity establishment and primordium initiation to specific genes. Investigation of roles of the same or homologous genes during leaf development has revealed strikingly similar functions in leaves compared to SAMs. Specifically, the marginal blastozone that characterizes many angiosperm leaves appears to function in a manner mechanistically similar to the SAM. We argue here that the similarities may be homologous due to descent from ancestral roles in an ancestral shoot system. Molecular aspects of SAM and leaf development in gymnosperms is largely neglected and could provide insight into seed plant leaf evolution.

Arabidopsis APETALA2 (AP2) controls seed mass maternally, with ap2 mutants producing larger seeds than wild type. Here, we show that AP2 influences development of the three major seed compartments: embryo, endosperm, and seed coat. AP2 appears to have a significant effect on endosperm development. ap2 mutant seeds undergo an extended period of rapid endosperm growth early in development relative to wild type. This early expanded growth period in ap2 seeds is associated with delayed endosperm cellularization and overgrowth of the endosperm central vacuole. The subsequent period of moderate endosperm growth is also extended in ap2 seeds largely due to persistent cell divisions at the endosperm periphery. The effect of AP2 on endosperm development is mediated by different mechanisms than parent-of-origin effects on seed size observed in interploidy crosses. Seed coat development is affected; integument cells of ap2 mutants are more elongated than wild type. We conclude that endosperm overgrowth and/or integument cell elongation create a larger postfertilization embryo sac into which the ap2 embryo can grow. Morphological development of the embryo is initially delayed in ap2 compared with wild-type seeds, but ap2 embryos become larger than wild type after the bent-cotyledon stage of development. ap2 embryos are able to fill the enlarged postfertilization embryo sac, because they undergo extended periods of cell proliferation and seed filling. We discuss potential mechanisms by which maternally acting AP2 influences development of the zygotic embryo and endosperm to repress seed size.

Leaves and stems are ultimately derived from the shoot apical meristem (SAM); leaves arise from the peripheral zone of the SAM and stem tissue is derived from both the peripheral and central zones of the SAM. Both the peripheral and central regions of the SAM are formed during embryogenesis when the basic body plan of the plant is established. Interplay between points of maximal concentration of auxin and specific patterns of transcription of both auxin-responsive transcription factors and other patterning genes subdivide the embryo along both the apical-basal and central-peripheral axes. Differential gene expression along these axes leads to the differentiation of tissues, lateral organs, meristems, and boundary regions, each with varying responsiveness to auxin. Subsequent shoot growth and development is a reiteration of basic patterning processes established during embryogenesis.

As more plant genome sequences become available, researchers are increasingly using comparative genomics to address some of the major questions in plant biology. Such questions include the evolution of photosynthesis and multicellularity, the developmental genetic changes responsible for alterations in body plan, and the origin of important plant innovations such as roots, leaves, and vascular tissue.

The origin of land plants from aquatic ancestors and their subsequent evolutionary radiation was due to major modifications in an ancestral developmental program allowing for plants of increasing complexity and stature. By comparing the developmental programs in different kinds of plants, we can understand how developmental differences lead to morphological differences. The ability to dissect the developmental genetic pathways of model angiosperm taxa such as Arabidopsis and maize has revealed many important developmental gene families that comprise the tool kit for flowering plant growth, patterning, and differentiation. This knowledge has been used to a limited extent as the basis for candidate gene approaches to explore the distribution and expression/function of a few developmental gene families in other flowering plants, gymnosperms, and to some extent, more distantly related plants. With the addition of nearly complete sequenced genomes for the moss Physcomitrella and the lycophyte Selaginella, comparisons of many more gene families are becoming possible. We performed searches of the unassembled Physcomitrella and Selaginella genomic sequences for some key developmental families. We combine these new data with information available in the literature from candidate gene approaches and present a first estimate of a suite of developmental genes that may have comprised the ancestral patterning tool kit of land plants. We conclude that the genome of the earliest embryophytes encoded homologues of many of the important developmental genes that have been identified in model angiosperm taxa, but the roles of these were probably somewhat different than the roles attributed to them in Arabidopsis and maize. Developmental gene families have diversified during land plant evolution, and genome complexity may correlate well with phylogenetic position.

Vascular plants diverged more than 400 million years ago into two lineages, the lycophytes and the euphyllophytes [1-4]. Leaf-like organs evolved independently in these two groups [1-3, 5, 6]. Microphylls in lycophytes are hypothesized to have originated as lateral outgrowths of tissue that later became vascularized (the enation theory) [2, 3, 5-8] or through the sterilization of sporangia (the sterilization hypothesis) [3]. Megaphylls in euphyllophytes are thought to represent modified lateral branches [7, 9, 10]. The fossil record also indicates that the seed plant megaphyll evolved uniquely in the ancestor of seed plants, independent of megaphylls in ferns, because seed plants evolved from leafless progymnosperm ancestors [3, 8, 11-13]. Surprisingly, a recent study of KNOX and ARP gene expression in a lycophyte was reported to indicate recruitment of a similar mechanism for determinacy in both types of leaves [14]. We examined the expression of Class III HD-Zip genes in the lycophyte Seiaginella kraussiana and in two gymnosperms, Ginkgo and Pseudotsuga. Our data indicate that mechanisms promoting leaf initiation, vascularization, and polarity are quite different in lycophytes and seed plants, consistent with the hypotheses that megaphylls originated as lateral branches whereas microphylls originated as tissue outgrowths.

Land plants underwent tremendous evolutionary change following the divergence of the ancestral lineage from algal relatives. Several important developmental innovations appeared as the embryophyte clade diversified, leading to the appearance of new organs and tissue types. To understand how these changes came about, we need to identify the fundamental genetic developmental programs that are responsible for growth, patterning, and differentiation and describe how these programs were modified and elaborated through time to produce novel morphologies. Class III homeodomain–leucine zipper (class III HD–Zip) genes, identified in the model plant Arabidopsis thaliana, provide good candidates for basic land plant patterning genes. We show that these genes may have evolved in a common ancestor of land plants and their algal sister group and that the gene family has diversified as land plant lineages have diversified. Phylogenetic analysis, expression data from nonflowering lineages, and evidence from Arabidopsis and other flowering plants indicate that class III HD–Zip genes acquired new functions in sporophyte apical growth, vascular patterning and differentiation, and leaf development. Modification of expression patterns that accompanied diversification of class III HD–Zip genes likely played an important role in the evolution of land plant form.

Small non-coding RNAs have been implicated in endogenous gene regulation and in the control of invading genetic entities. Small RNAs derived from double-stranded RNA that may or may not be encoded in the genome are termed short interfering RNAs (siRNAs). Such RNAs act to silence invading nucleic acids, such as viruses and transposons, via RNA cleavage or changes in chromatin structure. These phenomena have been termed posttranscriptional gene silencing (PTGS), virus-induced gene silencing (VIGS) and RNA interference (RNAi), and they function as a cellular or genomic immune system. In contrast, endogenously encoded small RNAs that are processed from stem-loop precursor RNAs and that regulate genes encoded at separate distinct loci are called microRNAs (miRNAs). These two classes of small RNA share similarities in their modes of action, suggesting similar biochemical complexes may mediate their function. However, they differ in both their biogenesis and their target sequences, with endogenous genes encoding miRNAs whose targets are other distinct endogenous genes and with siRNAs, whose targets are invading nucleic acids, being derived from double-stranded RNA due to either aberrant transcriptional patterns within transposons or production during viral infection. This chapter concentrates on the biogenesis, activity and evolution of miRNAs in plants, with reference to other systems when appropriate.