Complementing model species with model clades.

Model species continue to underpin groundbreaking plant science research. At the same time, the phylogenetic resolution of the land plant Tree of Life continues to improve. The intersection of these two research paths creates a unique opportunity to further extend the usefulness of model species across larger taxonomic groups. Here we promote the utility of the Arabidopsis thaliana model species, especially the ability to connect its genetic and functional resources, to species across the entire Brassicales order. We focus on the utility of using genomics and phylogenomics to bridge the evolution and diversification of several traits across the Brassicales to the resources in Arabidopsis, thereby extending scope from a model species by establishing a "model clade". These Brassicales-wide traits are discussed in the context of both the model species Arabidopsis thaliana and the family Brassicaceae. We promote the utility of such a "model clade" and make suggestions for building global networks to support future studies in the model order Brassicales.

BUZZ: an essential gene for postinitiation root hair growth and a mediator of root architecture in Brachypodium distachyon.

Here, we discover a player in root development. Recovered from a forward-genetic screen in Brachypodium distachyon, the buzz mutant initiates root hairs but they fail to elongate. In addition, buzz roots grow twice as fast as wild-type roots. Also, lateral roots show increased sensitivity to nitrate, whereas primary roots are less sensitive to nitrate. Using whole-genome resequencing, we identified the causal single nucleotide polymorphism as occurring in a conserved but previously uncharacterized cyclin-dependent kinase (CDK)-like gene. The buzz mutant phenotypes are rescued by the wild-type B. distachyon BUZZ coding sequence and by an apparent homolog in Arabidopsis thaliana. Moreover, T-DNA mutants in A. thaliana BUZZ have shorter root hairs. BUZZ mRNA localizes to epidermal cells and develops root hairs and, in the latter, partially colocalizes with the NRT1.1A nitrate transporter. Based on qPCR and RNA-Seq, buzz overexpresses ROOT HAIRLESS LIKE SIX-1 and -2 and misregulates genes related to hormone signaling, RNA processing, cytoskeletal, and cell wall organization, and to the assimilation of nitrate. Overall, these data demonstrate that BUZZ is required for tip growth after root hair initiation and root architectural responses to nitrate.

Annotation of the Turnera subulata (Passifloraceae) Draft Genome Reveals the S-Locus Evolved after the Divergence of Turneroideae from Passifloroideae in a Stepwise Manner.

A majority of Turnera species (Passifloraceae) exhibit distyly, a reproductive system involving both self-incompatibility and reciprocal herkogamy. This system differs from self-incompatibility in Passiflora species. The genetic basis of distyly in Turnera is a supergene, restricted to the S-morph, and containing three S-genes. How supergenes and distyly evolved in Turnera, and the other Angiosperm families exhibiting distyly remain largely unknown. Unraveling the evolutionary origins in Turnera requires the generation of genomic resources and extensive phylogenetic analyses. Here, we present the annotated draft genome of the S-morph of distylous Turnera subulata. Our annotation allowed for phylogenetic analyses of the three S-genes' families across 56 plant species ranging from non-seed plants to eudicots. In addition to the phylogenetic analysis, we identified the three S-genes' closest paralogs in two species of Passiflora. Our analyses suggest that the S-locus evolved after the divergence of Passiflora and Turnera. Finally, to provide insights into the neofunctionalization of the S-genes, we compared expression patterns of the S-genes with close paralogs in Arabidopsis and Populus trichocarpa. The annotation of the T. subulata genome will provide a useful resource for future comparative work. Additionally, this work has provided insights into the convergent nature of distyly and the origin of supergenes.

Plastid transit peptides-where do they come from and where do they all belong? Multi-genome and pan-genomic assessment of chloroplast transit peptide evolution.

Subcellular relocalization of proteins determines an organism's metabolic repertoire and thereby its survival in unique evolutionary niches. In plants, the plastid and its various morphotypes import a large and varied number of nuclear-encoded proteins to orchestrate vital biochemical reactions in a spatiotemporal context. Recent comparative genomics analysis and high-throughput shotgun proteomics data indicate that there are a large number of plastid-targeted proteins that are either semi-conserved or non-conserved across different lineages. This implies that homologs are differentially targeted across different species, which is feasible only if proteins have gained or lost plastid targeting peptides during evolution. In this study, a broad, multi-genome analysis of 15 phylogenetically diverse genera and in-depth analyses of pangenomes from Arabidopsis and Brachypodium were performed to address the question of how proteins acquire or lose plastid targeting peptides. The analysis revealed that random insertions or deletions were the dominant mechanism by which novel transit peptides are gained by proteins. While gene duplication was not a strict requirement for the acquisition of novel subcellular targeting, 40% of novel plastid-targeted genes were found to be most closely related to a sequence within the same genome, and of these, 30.5% resulted from alternative transcription or translation initiation sites. Interestingly, analysis of the distribution of amino acids in the transit peptides of known and predicted chloroplast-targeted proteins revealed monocot and eudicot-specific preferences in residue distribution.

Genome-Scale Characterization of Predicted Plastid-Targeted Proteomes in Higher Plants.

Plastids are morphologically and functionally diverse organelles that are dependent on nuclear-encoded, plastid-targeted proteins for all biochemical and regulatory functions. However, how plastid proteomes vary temporally, spatially, and taxonomically has been historically difficult to analyze at a genome-wide scale using experimental methods. A bioinformatics workflow was developed and evaluated using a combination of fast and user-friendly subcellular prediction programs to maximize performance and accuracy for chloroplast transit peptides and demonstrate this technique on the predicted proteomes of 15 sequenced plant genomes. Gene family grouping was then performed in parallel using modified approaches of reciprocal best BLAST hits (RBH) and UCLUST. A total of 628 protein families were found to have conserved plastid targeting across angiosperm species using RBH, and 828 using UCLUST. However, thousands of clusters were also detected where only one species had predicted plastid targeting, most notably in Panicum virgatum which had 1,458 proteins with species-unique targeting. An average of 45% overlap was found in plastid-targeted protein-coding gene families compared with Arabidopsis, but an additional 20% of proteins matched against the full Arabidopsis proteome, indicating a unique evolution of plastid targeting. Neofunctionalization through subcellular relocalization is known to impart novel biological functions but has not been described before on a genome-wide scale for the plastid proteome. Further work to correlate these predicted novel plastid-targeted proteins to transcript abundance and high-throughput proteomics will uncover unique aspects of plastid biology and shed light on how the plastid proteome has evolved to influence plastid morphology and biochemistry.

Co-expression clustering across flower development identifies modules for diverse floral forms in Achimenes (Gesneriaceae).

BACKGROUND: Genetic pathways involved with flower color and shape are thought to play an important role in the development of flowers associated with different pollination syndromes, such as those associated with bee, butterfly, or hummingbird pollination. Because pollination syndromes are complex traits that are orchestrated by multiple genes and pathways, the gene regulatory networks have not been explored. Gene co-expression networks provide a systems level approach to identify important contributors to floral diversification. METHODS: RNA-sequencing was used to assay gene expression across two stages of flower development (an early bud and an intermediate stage) in 10 species of Achimenes (Gesneriaceae). Two stage-specific co-expression networks were created from 9,503 orthologs and analyzed to identify module hubs and the network periphery. Module association with bee, butterfly, and hummingbird pollination syndromes was tested using phylogenetic mixed models. The relationship between network connectivity and evolutionary rates (d N/d S) was tested using linear models. RESULTS: Networks contained 65 and 62 modules that were largely preserved between developmental stages and contained few stage-specific modules. Over a third of the modules in both networks were associated with flower color, shape, and pollination syndrome. Within these modules, several hub nodes were identified that related to the production of anthocyanin and carotenoid pigments and the development of flower shape. Evolutionary rates were decreased in highly connected genes and elevated in peripheral genes. DISCUSSION: This study aids in the understanding of the genetic architecture and network properties underlying the development of floral form and provides valuable candidate modules and genes for future studies.

Genome-wide identification and analysis of the ALTERNATIVE OXIDASE gene family in diploid and hexaploid wheat.

A comprehensive understanding of wheat responses to environmental stress will contribute to the long-term goal of feeding the planet. ALERNATIVE OXIDASE (AOX) genes encode proteins involved in a bypass of the electron transport chain and are also known to be involved in stress tolerance in multiple species. Here, we report the identification and characterization of the AOX gene family in diploid and hexaploid wheat. Four genes each were found in the diploid ancestors Triticum urartu, and Aegilops tauschii, and three in Aegilops speltoides. In hexaploid wheat (Triticum aestivum), 20 genes were identified, some with multiple splice variants, corresponding to a total of 24 proteins for those with observed transcription and translation. These proteins were classified as AOX1a, AOX1c, AOX1e or AOX1d via phylogenetic analysis. Proteins lacking most or all signature AOX motifs were assigned to putative regulatory roles. Analysis of protein-targeting sequences suggests mixed localization to the mitochondria and other organelles. In comparison to the most studied AOX from Trypanosoma brucei, there were amino acid substitutions at critical functional domains indicating possible role divergence in wheat or grasses in general. In hexaploid wheat, AOX genes were expressed at specific developmental stages as well as in response to both biotic and abiotic stresses such as fungal pathogens, heat and drought. These AOX expression patterns suggest a highly regulated and diverse transcription and expression system. The insights gained provide a framework for the continued and expanded study of AOX genes in wheat for stress tolerance through breeding new varieties, as well as resistance to AOX-targeted herbicides, all of which can ultimately be used synergistically to improve crop yield.

Lessons from Cleomaceae, the Sister of Crucifers.

Cleomaceae is a diverse group well-suited to addressing fundamental genomic and evolutionary questions as the sister group to Brassicaceae, facilitating transfer of knowledge from the model Arabidopsis thaliana. Phylogenetic and taxonomic revisions provide a framework for examining the evolution of substantive morphological and physiology diversity in Cleomaceae, but not necessarily in Brassicaceae. The investigation of both nested and contrasting whole-genome duplications (WGDs) between Cleomaceae and Brassicaceae allows comparisons of independently duplicated genes and investigation of whether they may be drivers of the observed innovations. Further, a wealth of outstanding genetic research has provided insight into how the important alternative carbon fixation pathway, C4 photosynthesis, has evolved via differential expression of a suite of genes, of which the underlying mechanisms are being elucidated.

Non-dispersive phloem-protein bodies (NPBs) of Populus trichocarpa consist of a SEOR protein and do not respond to cell wounding and Ca2.

Differentiating sieve elements in the phloem of angiosperms produce abundant phloem-specific proteins before their protein synthesis machinery is degraded. These P-proteins initially form dense bodies, which disperse into individual filaments when the sieve element matures. In some cases, however, the dense protein agglomerations remain intact and are visible in functional sieve tubes as non-dispersive P-protein bodies, or NPBs. Species exhibiting NPBs are distributed across the entire angiosperm clade. We found that NPBs in the model tree, Populus trichocarpa, resemble the protein bodies described from other species of the order Malpighiales as they all consist of coaligned tubular fibrils bundled in hexagonal symmetry. NPBs of all Malpighiales tested proved unresponsive to sieve tube wounding and Ca2+. The P. trichocarpa NPBs consisted of a protein encoded by a gene that in the genome database of this species had been annotated as a homolog of SEOR1 (sieve element occlusion-related 1) in Arabidopsis. Sequencing of the gene in our plants corroborated this interpretation, and we named the gene PtSEOR1. Previously characterized SEOR proteins form irregular masses of P-protein slime in functional sieve tubes. We conclude that a subgroup of these proteins is involved in the formation of NPBs at least in the Malpighiales, and that these protein bodies have no role in rapid wound responses of the sieve tube network.

Phylogenomic analyses reveal extensive gene flow within the magic flowers (Achimenes).

PREMISE OF THE STUDY: The Neotropical Gesneriaceae is a lineage known for its colorful and diverse flowers, as well as an extensive history of intra- and intergeneric hybridization, particularly among Achimenes (the magic flowers) and other members of subtribe Gloxiniinae. Despite numerous studies seeking to elucidate the evolutionary relationships of these lineages, relatively few have sought to infer specific patterns of gene flow despite evidence of widespread hybridization. METHODS: To explore the utility of phylogenomic data for reassessing phylogenetic relationships and inferring patterns of gene flow among species of Achimenes, we sequenced 12 transcriptomes. We used a variety of methods to infer the species tree, examine gene tree discordance, and infer patterns of gene flow. KEY RESULTS: Phylogenomic analyses resolve clade relationships at the crown of the lineage with strong support. In contrast to previous analyses, we recovered strong support for several new relationships despite a significant amount of gene tree discordance. We present evidence for at least two introgression events between two species pairs that share pollinators, and suggest that the species status of Achimenes admirabilis be reexamined. CONCLUSIONS: Our study demonstrates the utility of transcriptome data for phylogenomic analyses, and inferring patterns of gene flow despite gene tree discordance. Moreover, these data provide another example of prevalent interspecific gene flow among Neotropical plants that share pollinators.

Comparative transcriptome analyses of flower development in four species of Achimenes (Gesneriaceae).

BACKGROUND: Flowers have an amazingly diverse display of colors and shapes, and these characteristics often vary significantly among closely related species. The evolution of diverse floral form can be thought of as an adaptive response to pollination and reproduction, but it can also be seen through the lens of morphological and developmental constraints. To explore these interactions, we use RNA-seq across species and development to investigate gene expression and sequence evolution as they relate to the evolution of the diverse flowers in a group of Neotropical plants native to Mexico-magic flowers (Achimenes, Gesneriaceae). RESULTS: The assembled transcriptomes contain between 29,000 and 42,000 genes expressed during development. We combine sequence orthology and coexpression clustering with analyses of protein evolution to identify candidate genes for roles in floral form evolution. Over 25% of transcripts captured were distinctive to Achimenes and overrepresented by genes involved in transcription factor activity. Using a model-based clustering approach we find dynamic, temporal patterns of gene expression among species. Selection tests provide evidence of positive selection in several genes with roles in pigment production, flowering time, and morphology. Combining these approaches to explore genes related to flower color and flower shape, we find distinct patterns that correspond to transitions of floral form among Achimenes species. CONCLUSIONS: The floral transcriptomes developed from four species of Achimenes provide insight into the mechanisms involved in the evolution of diverse floral form among closely related species with different pollinators. We identified several candidate genes that will serve as an important and useful resource for future research. High conservation of sequence structure, patterns of gene coexpression, and detection of positive selection acting on few genes suggests that large phenotypic differences in floral form may be caused by genetic differences in a small set of genes. Our characterized floral transcriptomes provided here should facilitate further analyses into the genomics of flower development and the mechanisms underlying the evolution of diverse flowers in Achimenes and other Neotropical Gesneriaceae.

Distinct Processes Drive Diversification in Different Clades of Gesneriaceae.

Using a time-calibrated phylogenetic hypothesis including 768 Gesneriaceae species (out of [Formula: see text]3300 species) and more than 29,000 aligned bases from 26 gene regions, we test Gesneriaceae for diversification rate shifts and the possible proximal drivers of these shifts: geographic distributions, growth forms, and pollination syndromes. Bayesian Analysis of Macroevolutionary Mixtures analyses found five significant rate shifts in Beslerieae, core Nematanthus, core Columneinae, core Streptocarpus, and Pacific Cyrtandra These rate shifts correspond with shifts in diversification rates, as inferred by Binary State Speciation and Extinction Model and Geographic State Speciation and Extinction model, associated with hummingbird pollination, epiphytism, unifoliate growth, and geographic area. Our results suggest that diversification processes are extremely variable across Gesneriaceae clades with different combinations of characters influencing diversification rates in different clades. Diversification patterns between New and Old World lineages show dramatic differences, suggesting that the processes of diversification in Gesneriaceae are very different in these two geographic regions.

Kranz and single-cell forms of C4 plants in the subfamily Suaedoideae show kinetic C4 convergence for PEPC and Rubisco with divergent amino acid substitutions.

The two carboxylation reactions performed by phosphoenolpyruvate carboxylase (PEPC) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) are vital in the fixation of inorganic carbon for C4 plants. The abundance of PEPC is substantially elevated in C4 leaves, while the location of Rubisco is restricted to one of two chloroplast types. These differences compared with C3 leaves have been shown to result in convergent enzyme optimization in some C4 species. Investigation into the kinetic properties of PEPC and Rubisco from Kranz C4, single cell C4, and C3 species in Chenopodiaceae s. s. subfamily Suaedoideae showed that these major carboxylases in C4 Suaedoideae species lack the same mutations found in other C4 systems which have been examined; but still have similar convergent kinetic properties. Positive selection analysis on the N-terminus of PEPC identified residues 364 and 368 to be under positive selection with a posterior probability >0.99 using Bayes empirical Bayes. Compared with previous analyses on other C4 species, PEPC from C4 Suaedoideae species have different convergent amino acids that result in a higher K m for PEP and malate tolerance compared with C3 species. Kinetic analysis of Rubisco showed that C4 species have a higher catalytic efficiency of Rubisco (k catc in mol CO2 mol(-1) Rubisco active sites s(-1)), despite lacking convergent substitutions in the rbcL gene. The importance of kinetic changes to the two-carboxylation reactions in C4 leaves related to amino acid selection is discussed.

Environmental and genetic correlates of allocation to sexual reproduction in the circumpolar plant Bistorta vivipara.

UNLABELLED: • PREMISE OF THE STUDY: Sexual reproduction often requires more energy and time than clonal reproduction. In marginal arctic conditions, species that can reproduce both sexually and clonally dominate. Plants with this capacity may thrive because they can alter reproduction depending on environmental conditions. Bistorta vivipara is a circumpolar herb that predominately reproduces clonally, but certain environmental conditions promote higher investment in flowers (and possible sexual reproduction). Despite largely reproducing clonally, the herb has high levels of genetic variation, and the processes underlying this paradoxical pattern of variation remain unclear. Here we identified environmental factors associated with sexual investment and examined whether sexual reproduction is associated with higher levels of genetic variation.• METHODS: We sampled 20 populations of B. vivipara across the high Arctic archipelago of Svalbard. In each population, we measured reproductive traits, environmental variables, and collected samples for genetic analyses. These samples permitted hypotheses to be tested regarding sexual investment and ecological and genetic correlates.• KEY RESULTS: Increased soil nitrogen and organic matter content and decreased elevation were positively associated with investment in flowers. Increased investment in flowers significantly correlated with more genotypes per population. Linkage disequilibrium was consistent with predominant clonality, but several populations showed higher genetic variation and lower differentiation than expected. There was no geographical genetic structure.• CONCLUSIONS: In B. vivipara, sexual investment is positively associated with habitat quality. Bistorta vivipara predominantly reproduces clonally, but occasional outcrossing, efficient clonal reproduction, and dispersal by bulbils can explain the considerable genetic variation and weak genetic structure in B. vivipara.

Insights into the evolution and diversification of the AT-hook Motif Nuclear Localized gene family in land plants.

BACKGROUND: Members of the ancient land-plant-specific transcription factor AT-Hook Motif Nuclear Localized (AHL) gene family regulate various biological processes. However, the relationships among the AHL genes, as well as their evolutionary history, still remain unexplored. RESULTS: We analyzed over 500 AHL genes from 19 land plant species, ranging from the early diverging Physcomitrella patens and Selaginella to a variety of monocot and dicot flowering plants. We classified the AHL proteins into three types (Type-I/-II/-III) based on the number and composition of their functional domains, the AT-hook motif(s) and PPC domain. We further inferred their phylogenies via Bayesian inference analysis and predicted gene gain/loss events throughout their diversification. Our analyses suggested that the AHL gene family emerged in embryophytes and further evolved into two distinct clades, with Type-I AHLs forming one clade (Clade-A), and the other two types together diversifying in another (Clade-B). The two AHL clades likely diverged before the separation of Physcomitrella patens from the vascular plant lineage. In angiosperms, Clade-A AHLs expanded into 5 subfamilies; while, the ones in Clade-B expanded into 4 subfamilies. Examination of their expression patterns suggests that the AHLs within each clade share similar expression patterns with each other; however, AHLs in one monophyletic clade exhibit distinct expression patterns from the ones in the other clade. Over-expression of a Glycine max AHL PPC domain in Arabidopsis thaliana recapitulates the phenotype observed when over-expressing its Arabidopsis thaliana counterpart. This result suggests that the AHL genes from different land plant species may share conserved functions in regulating plant growth and development. Our study further suggests that such functional conservation may be due to conserved physical interactions among the PPC domains of AHL proteins. CONCLUSIONS: Our analyses reveal a possible evolutionary scenario for the AHL gene family in land plants, which will facilitate the design of new studies probing their biological functions. Manipulating the AHL genes has been suggested to have tremendous effects in agriculture through increased seedling establishment, enhanced plant biomass and improved plant immunity. The information gleaned from this study, in turn, has the potential to be utilized to further improve crop production.

Positive selection of Kranz and non-Kranz C4 phosphoenolpyruvate carboxylase amino acids in Suaedoideae (Chenopodiaceae).

In subfamily Suaedoideae, four independent gains of C4 photosynthesis are proposed, which includes two parallel origins of Kranz anatomy (sections Salsina and Schoberia) and two independent origins of single-cell C4 anatomy (Bienertia and Suaeda aralocaspica). Additional phylogenetic support for this hypothesis was generated from sequence data of the C-terminal portion of the phosphoenolpyruvate carboxylase (PEPC) gene used in C4 photosynthesis (ppc-1) in combination with previous sequence data. ppc-1 sequence was generated for 20 species in Suaedoideae and two outgroup Salsola species that included all types of C4 anatomies as well as two types of C3 anatomies. A branch-site test for positively selected codons was performed using the software package PAML. From labelling of the four branches where C4 is hypothesized to have developed (foreground branches), residue 733 (maize numbering) was identified to be under positive selection with a posterior probability >0.99 and residue 868 at the >0.95 interval using Bayes empirical Bayes (BEB). When labelling all the branches within C4 clades, the branch-site test identified 13 codons to be under selection with a posterior probability >0.95 by BEB; this is discussed considering current information on functional residues. The signature C4 substitution of an alanine for a serine at position 780 in the C-terminal end (which is considered a major determinant of affinity for PEP) was only found in four of the C4 species sampled, while eight of the C4 species and all the C3 species have an alanine residue; indicating that this substitution is not a requirement for C4 function.

Phylogenetic relationships among the North American cleomoids (Cleomaceae): a test of Iltis's reduction series.

PREMISE OF STUDY: A monophyletic group composed of five genera of the Cleomaceae represents an intriguing lineage with outstanding taxonomic and evolutionary questions. Generic boundaries are poorly defined, and historical hypotheses regarding the evolution of fruit type and phylogenetic relationships provide testable questions. This is the first detailed phylogenetic investigation of all 22 species in this group. We use this phylogenetic framework to assess generic monophyly and test Iltis's evolutionary "reduction series" hypothesis regarding phylogeny and fruit type/seed number. • METHODS: Maximum likelihood and Bayesian analyses of four plastid intergenic spacer region sequences (rpl32-trnL, trnQ-rps16, ycf1-rps15, and psbA-trnH) and one nuclear (ITS) region were used to reconstruct phylogenetic relationships among the NA cleomoid species. Stochastic mapping and ancestral-state reconstruction were used to study the evolution of fruit type. • KEY RESULTS: Both analyses recovered nearly identical phylogenies. Three of the currently recognized genera (Wislizenia, Carsonia, and Oxystylis) are monophyletic while two (Cleomella and Peritoma) are para- or polyphyletic. There was a single origin of the two-seeded schizocarp in the ancestor of the Oxystylis-Wislizenia clade and a secondary derivation of elongated capsule-type fruits in Peritoma from a truncated capsule state in Cleomella. • CONCLUSIONS: Our well-resolved phylogeny supports most of the current species circumscriptions but not current generic circumscriptions. Additionally, our results are inconsistent with Iltis's hypothesis of species with elongated many-seed fruits giving rise to species with truncated few-seeded fruits. Instead, we find support for the reversion to elongated multiseeded fruits from a truncate few-seeded ancestor in Peritoma.

Structural and physiological analyses in Salsoleae (Chenopodiaceae) indicate multiple transitions among C3, intermediate, and C4 photosynthesis.

In subfamily Salsoloideae (family Chenopodiaceae) most species are C4 plants having terete leaves with Salsoloid Kranz anatomy characterized by a continuous dual chlorenchyma layer of Kranz cells (KCs) and mesophyll (M) cells, surrounding water storage and vascular tissue. From section Coccosalsola sensu Botschantzev, leaf structural and photosynthetic features were analysed on selected species of Salsola which are not performing C4 based on leaf carbon isotope composition. The results infer the following progression in distinct functional and structural forms from C3 to intermediate to C4 photosynthesis with increased leaf succulence without changes in vein density: From species performing C3 photosynthesis with Sympegmoid anatomy with two equivalent layers of elongated M cells, with few organelles in a discontinuous layer of bundle sheath (BS) cells (S. genistoides, S. masenderanica, S. webbii) > development of proto-Kranz BS cells having mitochondria in a centripetal position and increased chloroplast number (S. montana) > functional C3-C4 intermediates having intermediate CO2 compensation points with refixation of photorespired CO2, development of Kranz-like anatomy with reduction in the outer M cell layer to hypodermal-like cells, and increased specialization (but not size) of a Kranz-like inner layer of cells with increased cell wall thickness, organelle number, and selective expression of mitochondrial glycine decarboxylase (Kranz-like Sympegmoid, S. arbusculiformis; and Kranz-like Salsoloid, S. divaricata) > selective expression of enzymes between the two cell types for performing C4 with Salsoloid-type anatomy. Phylogenetic analysis of tribe Salsoleae shows the occurrence of C3 and intermediates in several clades, and lineages of interest for studying different forms of anatomy.

Antisense expression of peach mildew resistance locus O (PpMlo1) gene confers cross-species resistance to powdery mildew in Fragaria x ananassa.

Powdery mildew (PM) is one of the major plant pathogens. The conventional method of PM control includes frequent use of sulfur-based fungicides adding to production costs and potential harm to the environment. PM remains a major scourge for Rosaceae crops where breeding approaches mainly resort to gene-for-gene resistance. We have tested an alternate source of PM resistance in Rosaceae. Mildew resistance locus O (MLO) has been well studied in barley due to its role in imparting broad spectrum resistance to PM. We identified PpMlo1 (Prunus persica Mlo) in peach and characterized it further to test if a similar mechanism of resistance is conserved in Rosaceae. Due to its recalcitrance in tissue culture, reverse genetic studies involving PpMloI were not feasible in peach. Therefore, Fragaria x ananassa LF9 line, a taxonomic surrogate, was used for functional analysis of PpMlo1. Agrobacterium-mediated transformation yielded transgenic strawberry plants expressing PpMlo1 in sense and antisense orientation. Antisense expression of PpMlo1 in transgenic strawberry plants conferred resistance to Fragaria-specific powdery mildew, Podosphaera macularis. Phylogenetic analysis of 208 putative Mlo gene copies from 35 plant species suggests a large number of duplications of this gene family prior to the divergence of monocots and eudicots, early in eudicot diversification. Our results indicate that the Mlo-based resistance mechanism is functional in Rosaceae, and that Fragaria can be used as a host to test mechanistic function of genes derived from related tree species. To the best of our knowledge, this work is one of the first attempts at testing the potential of using a Mlo-based resistance strategy to combat powdery mildew in Rosaceae.