Pervasive G × E interactions shape adaptive trajectories and the exploration of the phenotypic space in artificial selection experiments.

Quantitative genetics models have shown that long-term selection responses depend on initial variance and mutational influx. Understanding limits of selection requires quantifying the role of mutational variance. However, correlative responses to selection on nonfocal traits can perturb the selection response on the focal trait; and generations are often confounded with selection environments so that genotype by environment (G×E) interactions are ignored. The Saclay divergent selection experiments (DSEs) on maize flowering time were used to track the fate of individual mutations combining genotyping data and phenotyping data from yearly measurements (DSEYM) and common garden experiments (DSECG) with four objectives: (1) to quantify the relative contribution of standing and mutational variance to the selection response, (2) to estimate genotypic mutation effects, (3) to study the impact of G×E interactions in the selection response, and (4) to analyze how trait correlations modulate the exploration of the phenotypic space. We validated experimentally the expected enrichment of fixed beneficial mutations with an average effect of +0.278 and +0.299 days to flowering, depending on the genetic background. Fixation of unfavorable mutations reached up to 25% of incoming mutations, a genetic load possibly due to antagonistic pleiotropy, whereby mutations fixed in the selection environment (DSEYM) turned to be unfavorable in the evaluation environment (DSECG). Global patterns of trait correlations were conserved across genetic backgrounds but exhibited temporal patterns. Traits weakly or uncorrelated with flowering time triggered stochastic exploration of the phenotypic space, owing to microenvironment-specific fixation of standing variants and pleiotropic mutational input.

Evolutionary analyses and expression patterns of TCP genes in Ranunculales.

TCP transcription factors play a role in a large number of developmental processes and are at the crossroads of numerous hormonal biosynthetic and signaling pathways. The complete repertoire of TCP genes has already been characterized in several plant species, but not in any species of early diverging eudicots. We focused on the order Ranunculales because of its phylogenetic position as sister group to all other eudicots and its important morphological diversity. Results show that all the TCP genes expressed in the floral transcriptome of Nigella damascena (Ranunculaceae) are the orthologs of the TCP genes previously identified from the fully sequenced genome of Aquilegia coerulea. Phylogenetic analyses combined with the identification of conserved amino acid motifs suggest that six paralogous genes of class I TCP transcription factors were present in the common ancestor of angiosperms. We highlight independent duplications in core eudicots and Ranunculales within the class I and class II subfamilies, resulting in different numbers of paralogs within the main subclasses of TCP genes. This has most probably major consequences on the functional diversification of these genes in different plant clades. The expression patterns of TCP genes in Nigella damascena were consistent with the general suggestion that CIN and class I TCP genes may have redundant roles or take part in same pathways, while CYC/TB1 genes have more specific actions. Our findings open the way for future studies at the tissue level, and for investigating redundancy and subfunctionalisation in TCP genes and their role in the evolution of morphological novelties.

Micro- and Macroscale Patterns of Petal Morphogenesis in Nigella damascena (Ranunculaceae) Revealed by Geometric Morphometrics and Cellular Analyses.

Petals, the inner organs in a differentiated perianth, generally play an important role in pollinator attraction. As such they exhibit an extraordinary diversity of shapes, sizes, and colors. Being involved in pollinator attraction and reward, they are privileged targets of evolution. The corolla of the Ranunculaceae species Nigella damascena consists of elaborate nectariferous petals, made of a stalk, upper, and lower lips forming a nectar pouch, shiny pseudonectaries, and pilose ears. While the main events of petal development are properly described, a few is known about the pattern of organ size and shape covariation and the cellular dynamics during development. In this study, we investigated the relationships between morphogenesis and growth of N. damascena petals using geometric morphometrics coupled with the study of cell characteristics. First, we found that petal shape and size dynamics are allometric during development and that their covariation suggests that petal shape change dynamics are exponentially slower than growth. We then found that cell proliferation is the major driver of shape patterning during development, while petal size dynamics are mostly driven by cell expansion. Our analyses provide a quantitative basis to characterize the relationships between shape, size, and cell characteristics during the development of an elaborate floral structure. Such studies lay the ground for future evo-devo investigations of the large morphological diversity observed in nectariferous structures, in Ranunculaceae and beyond.

Transcriptome Analysis Reveals Putative Target Genes of APETALA3-3 During Early Floral Development in Nigella damascena L.

Even though petals are homoplastic structures, their identity consistently involves genes of the APETALA3 (AP3) lineage. However, the extent to which the networks downstream of AP3 are conserved in species with petals of different evolutionary origins is unknown. In Ranunculaceae, the specificity of the AP3-III lineage offers a great opportunity to identify the petal gene regulatory network in a comparative framework. Using a transcriptomic approach, we investigated putative target genes of the AP3-III ortholog NdAP3-3 in Nigella damascena at early developmental stages when petal identity is determined, and we compared our data with that from selected eudicot species. We generated a de novo reference transcriptome to carry out a differential gene expression analysis between the wild-type and mutant NdAP3-3 genotypes differing by the presence vs. absence of petals at early stages of floral development. Among the 1,620 genes that were significantly differentially expressed between the two genotypes, functional annotation suggested a large involvement of nuclear activities, including regulation of transcription, and enrichment in processes linked to cell proliferation. Comparing with Arabidopsis data, we found that highly conserved genes between the two species are enriched in homologs of direct targets of the AtAP3 protein. Integrating AP3-3 binding site data from another Ranunculaceae species, Aquilegia coerulea, allowed us to identify a set of 18 putative target genes that were conserved between the three species. Our results suggest that, despite the independent evolutionary origin of petals in core eudicots and Ranunculaceae, a small conserved set of genes determines petal identity and early development in these taxa.

Unraveling the Developmental and Genetic Mechanisms Underpinning Floral Architecture in Proteaceae.

Proteaceae are a basal eudicot family with a highly conserved floral groundplan but which displays considerable variation in other aspects of floral and inflorescence morphology. Their morphological diversity and phylogenetic position make them good candidates for understanding the evolution of floral architecture, in particular the question of the homology of the undifferentiated perianth with the differentiated perianth of core eudicots, and the mechanisms underlying the repeated evolution of zygomorphy. In this paper, we combine a morphological approach to explore floral ontogenesis and a transcriptomic approach to access the genes involved in floral organ identity and development, focusing on Grevillea juniperina, a species from subfamily Grevilleoideae. We present developmental data for Grevillea juniperina and three additional species that differ in their floral symmetry using stereomicroscopy, SEM and High Resolution X-Ray Computed Tomography. We find that the adnation of stamens to tepals takes place at early developmental stages, and that the establishment of bilateral symmetry coincides with the asymmetrical growth of the single carpel. To set a framework for understanding the genetic basis of floral development in Proteaceae, we generated and annotated de novo a reference leaf/flower transcriptome from Grevillea juniperina. We found Grevillea homologs of all lineages of MADS-box genes involved in floral organ identity. Using Arabidopsis thaliana gene expression data as a reference, we found homologs of other genes involved in floral development in the transcriptome of G. juniperina. We also found at least 21 class I and class II TCP genes, a gene family involved in the regulation of growth processes, including floral symmetry. The expression patterns of a set of floral genes obtained from the transcriptome were characterized during floral development to assess their organ specificity and asymmetry of expression.

Characterization of CYCLOIDEA-like genes in Proteaceae, a basal eudicot family with multiple shifts in floral symmetry.

BACKGROUND AND AIMS: The basal eudicot family Proteaceae (approx. 1700 species) shows considerable variation in floral symmetry but has received little attention in studies of evolutionary development at the genetic level. A framework for understanding the shifts in floral symmetry in Proteaceae is provided by reconstructing ancestral states on an upated phylogeny of the family, and homologues of CYCLOIDEA (CYC), a key gene for the control of floral symmetry in both monocots and eudicots, are characterized. METHODS: Perianth symmetry transitions were reconstructed on a new species-level tree using parsimony and maximum likelihood. CYC-like genes in 35 species (31 genera) of Proteaceae were sequenced and their phylogeny was reconstructed. Shifts in selection pressure following gene duplication were investigated using nested branch-site models of sequence evolution. Expression patterns of CYC homologues were characterized in three species of Grevillea with different types of floral symmetry. KEY RESULTS: Zygomorphy has evolved 10-18 times independently in Proteaceae from actinomorphic ancestors, with at least four reversals to actinomorphy. A single duplication of CYC-like genes occurred prior to the diversification of Proteaceae, with putative loss or divergence of the ProtCYC1 paralogue in more than half of the species sampled. No shifts in selection pressure were detected in the branches subtending the two ProtCYC paralogues. However, the amino acid sequence preceding the TCP domain is strongly divergent in Grevillea ProtCYC1 compared with other species. ProtCYC genes were expressed in developing flowers of both actinomorphic and zygomorphic Grevillea species, with late asymmetric expression in the perianth of the latter. CONCLUSION: Proteaceae is a remarkable family in terms of the number of transitions in floral symmetry. Furthermore, although CYC-like genes in Grevillea have unusual sequence characteristics, they display patterns of expression that make them good candidates for playing a role in the establishment of floral symmetry.

Specific duplication and dorsoventrally asymmetric expression patterns of Cycloidea-like genes in zygomorphic species of Ranunculaceae.

Floral bilateral symmetry (zygomorphy) has evolved several times independently in angiosperms from radially symmetrical (actinomorphic) ancestral states. Homologs of the Antirrhinum majus Cycloidea gene (Cyc) have been shown to control floral symmetry in diverse groups in core eudicots. In the basal eudicot family Ranunculaceae, there is a single evolutionary transition from actinomorphy to zygomorphy in the stem lineage of the tribe Delphinieae. We characterized Cyc homologs in 18 genera of Ranunculaceae, including the four genera of Delphinieae, in a sampling that represents the floral morphological diversity of this tribe, and reconstructed the evolutionary history of this gene family in Ranunculaceae. Within each of the two RanaCyL (Ranunculaceae Cycloidea-like) lineages previously identified, an additional duplication possibly predating the emergence of the Delphinieae was found, resulting in up to four gene copies in zygomorphic species. Expression analyses indicate that the RanaCyL paralogs are expressed early in floral buds and that the duration of their expression varies between species and paralog class. At most one RanaCyL paralog was expressed during the late stages of floral development in the actinomorphic species studied whereas all paralogs from the zygomorphic species were expressed, composing a species-specific identity code for perianth organs. The contrasted asymmetric patterns of expression observed in the two zygomorphic species is discussed in relation to their distinct perianth architecture.

Combining phylogenetic and syntenic analyses for understanding the evolution of TCP ECE genes in eudicots.

TCP ECE genes encode transcription factors which have received much attention for their repeated recruitment in the control of floral symmetry in core eudicots, and more recently in monocots. Major duplications of TCP ECE genes have been described in core eudicots, but the evolutionary history of this gene family is unknown in basal eudicots. Reconstructing the phylogeny of ECE genes in basal eudicots will help set a framework for understanding the functional evolution of these genes. TCP ECE genes were sequenced in all major lineages of basal eudicots and Gunnera which belongs to the sister clade to all other core eudicots. We show that in these lineages they have a complex evolutionary history with repeated duplications. We estimate the timing of the two major duplications already identified in the core eudicots within a timeframe before the divergence of Gunnera and after the divergence of Proteales. We also use a synteny-based approach to examine the extent to which the expansion of TCP ECE genes in diverse eudicot lineages may be due to genome-wide duplications. The three major core-eudicot specific clades share a number of collinear genes, and their common evolutionary history may have originated at the γ event. Genomic comparisons in Arabidopsis thaliana and Solanumlycopersicum highlight their separate polyploid origin, with syntenic fragments with and without TCP ECE genes showing differential gene loss and genomic rearrangements. Comparison between recently available genomes from two basal eudicots Aquilegiacoerulea and Nelumbonucifera suggests that the two TCP ECE paralogs in these species are also derived from large-scale duplications. TCP ECE loci from basal eudicots share many features with the three main core eudicot loci, and allow us to infer the makeup of the ancestral eudicot locus.

Asymmetric morphogenetic cues along the transverse plane: shift from disymmetry to zygomorphy in the flower of Fumarioideae.

PREMISE OF THE STUDY: Zygomorphy has evolved multiple times in angiosperms. Near-actinomorphy is the ancestral state in the early diverging eudicot family Papaveraceae. Zygomorphy evolved once in the subfamily Fumarioideae from a disymmetric state. Unusual within angiosperms, zygomorphy takes place along the transverse plane of the flower. METHODS: We investigated floral development to understand the developmental bases of the evolution of floral symmetry in Papaveraceae. We then assessed the expression of candidate genes for the key developmental events responsible for the shift from disymmetry to transverse zygomorphy, namely CrabsClaw for nectary formation (PapCRC), ShootMeristemless (PapSTL) for spur formation, and Cycloidea (PapCYL) for growth control. KEY RESULTS: We found that an early disymmetric groundplan is common to all species studied, and that actinomorphy was acquired after sepal initiation in Papaveroideae. The shift from disymmetry to zygomorphy in Fumarioideae was associated with early asymmetric growth of stamen filaments, followed by asymmetric development of nectary outgrowth and spur along the transverse plane. Patterns of PapSTL expression could not be clearly related to spur formation. PapCRC and PapCYL genes were expressed in the nectary outgrowths, with a pattern of expression correlated with asymmetric nectary development in the zygomorphic species. Additionally, PapCYL genes were found asymmetrically expressed along the transverse plane in the basal region of outer petals in the zygomorphic species. CONCLUSION: Genes of PapCRC and PapCYL families could be direct or indirect targets of the initial transversally asymmetric cue responsible for the shift from disymmetry to zygomorphy in Fumarioideae.

Developmental analysis of maize endosperm proteome suggests a pivotal role for pyruvate orthophosphate dikinase.

Although the morphological steps of maize (Zea mays) endosperm development are well described, very little is known concerning the coordinated accumulation of the numerous proteins involved. Here, we present a proteomic study of maize endosperm development. The accumulation pattern of 409 proteins at seven developmental stages was examined. Hierarchical clustering analysis allowed four main developmental profiles to be recognized. Comprehensive investigation of the functions associated with clusters resulted in a consistent picture of the developmental coordination of cellular processes. Early stages, devoted to cellularization, cell division, and cell wall deposition, corresponded to maximal expression of actin, tubulins, and cell organization proteins, of respiration metabolism (glycolysis and tricarboxylic acid cycle), and of protection against reactive oxygen species. An important protein turnover, which is likely associated with the switch from growth and differentiation to storage, was also suggested from the high amount of proteases. A relative increase of abundance of the glycolytic enzymes compared to tricarboxylic acid enzymes is consistent with the recent demonstration of anoxic conditions during starch accumulation in the endosperm. The specific late-stage accumulation of the pyruvate orthophosphate dikinase may suggest a critical role of this enzyme in the starch-protein balance through inorganic pyrophosphate-dependent restriction of ADP-glucose synthesis in addition to its usually reported influence on the alanine-aromatic amino acid synthesis balance.

Diversity and evolution of CYCLOIDEA-like TCP genes in relation to flower development in Papaveraceae.

Monosymmetry evolved several times independently during flower evolution. In snapdragon (Antirrhinum majus), a key gene for monosymmetry is CYCLOIDEA (CYC), which belongs to the class II TCP gene family encoding transcriptional activators. We address the questions of the evolutionary history of this gene family and of possible recruitment of genes homologous to CYC in floral development and symmetry in the Papaveraceae. Two to three members of the class II TCP family were found in each species analyzed, two of which were CYC-like genes, on the basis of the presence of both the TCP and R conserved domains. The duplication that gave rise to these two paralogous lineages (named PAPACYL1 and PAPACYL2) probably predates the divergence of the two main clades within the Papaveraceae. Phylogenetic relationships among angiosperm class II TCP genes indicated that (1) PAPACYL genes were closest to Arabidopsis (Arabidopsis thaliana) AtTCP18, and a duplication at the base of the core eudicot would have given rise to two supplementary CYC-like lineages; and (2) at least three class II TCP genes were present in the ancestor of monocots and eudicots. Semiquantitative reverse transcription-polymerase chain reaction and in situ hybridization approaches in three species with different floral symmetry indicated that both PAPACYL paralogs were expressed during floral development. A pattern common to all three species was observed at organ junctions in inflorescences and flowers. Expression in the outer petals was specifically observed in the two species with nonactinomorphic flowers. Hypotheses concerning the ancestral pattern of expression and function of CYC-like genes and their possible role in floral development of Papaveraceae species leading to bisymmetric buds are discussed.

QTLs for enzyme activities and soluble carbohydrates involved in starch accumulation during grain filling in maize.

ADPglucose, the essential substrate for starch synthesis, is synthesized in maize by a pathway involving at least invertases, sucrose synthase, and ADPglucose pyrophosphorylase, as shown by the starch-deficient mutants, mn1, sh1, and bt2 or sh2, respectively. To improve understanding of the relationship between early grain-filling traits and carbohydrate composition in mature grain, QTLs linked to soluble invertase, sucrose synthase, and ADPglucose pyrophosphorylase activities and to starch, sucrose, fructose, and glucose concentrations were investigated. In order to take into account the specific time-course of each enzyme activity during grain filling, sampling was carried out at three periods (15, 25, and 35 d after pollination) on 100 lines from a recombinant inbred family, grown in the field. The MQTL method associated with QTL interaction analysis revealed numerous QTLs for all traits, but only one QTL was consistently observed at the three sampling periods. Some chromosome zones were heavily labelled, forming clusters of QTLs. Numerous possible candidate genes of the starch synthetic pathway co-located with QTLs. Four QTLs were found close to the locus Sh1 (bin 9.01) coding for the sucrose synthase. In order to confirm the importance of this locus, the CAPS polymorphism of the Sh1 gene was analysed in 45 genetically unrelated maize lines from various geographical origins. The DNA polymorphism was significantly associated with phenotypic traits related to grain filling (starch and amylose content, grain matter, and ADPglucose pyrophosphorylase activity at 35 DAP). Thus, the Sh1 locus could provide a physiologically pertinent marker for maize selection.

Regulation of lysine metabolism and endosperm protein synthesis by the opaque-5 and opaque-7 maize mutations.

Two high lysine maize endosperm mutations, opaque-5 (o5) and opaque-7 (o7), were biochemically characterized for endosperm protein synthesis and lysine metabolism in immature seeds. Albumins, globulins, and glutelins, which have a high content of lysine, were shown to be increased in the mutants, whereas zeins, which contain trace concentrations of lysine, were reduced in relation to the wild-type lines B77xB79+ and B37+. These alterations in the storage protein fraction distribution possibly explain the increased concentration of lysine in the two mutants. Using two-dimensional polyacrylamide gel electrophoresis of proteins of mature grains, variable amounts of zein polypeptides were detected and considerable differences were noted between the four lines studied. The analysis of the enzymes involved in lysine metabolism indicated that both mutants have reduced lysine catabolism when compared to their respective wild types, thus allowing more lysine to be available for storage protein synthesis.

Regulation of maize lysine metabolism and endosperm protein synthesis by opaque and floury mutations.

The capacity of two maize opaque endosperm mutants (o1 and o2) and two floury (fl1 and fl2) to accumulate lysine in the seed in relation to their wild type counterparts Oh43+ was examined. The highest total lysine content was 3.78% in the o2 mutant and the lowest 1.87% in fl1, as compared with the wild type (1.49%). For soluble lysine, o2 exhibited over a 700% increase, whilst for fl3 a 28% decrease was encountered, as compared with the wild type. In order to understand the mechanisms causing these large variations in both total and soluble lysine content, a quantitative and qualitative study of the N constituents of the endosperm has been carried out and data obtained for the total protein, nonprotein N, soluble amino acids, albumins/globulins, zeins and glutelins present in the seed of the mutants. Following two-dimensional PAGE separation, a total of 35 different forms of zein polypeptides were detected and considerable differences were noted between the five different lines. In addition, two enzymes of the aspartate biosynthetic pathway, aspartate kinase and homoserine dehydrogenase were analyzed with respect to feedback inhibition by lysine and threonine. The activities of the enzymes lysine 2-oxoglutate reductase and saccharopine dehydrogenase, both involved in lysine degradation in the maize endosperm were also determined and shown to be reduced several fold with the introduction of the o2, fl1 and fl2 mutations in the Oh43+ inbred line, whereas wild-type activity levels were verified in the Oh43o1 mutant.

An efficient solubilization buffer for plant proteins focused in immobilized pH gradients.

The solubilization of a large array of proteins before electrophoresis itself is a very critical point for proteomic analyses. We compared the efficiency of several different solubilization buffers. From this work, we defined a very efficient solubilization buffer, including two chaotropes, two reducing agents (R2), two detergents (D2), and two kinds of carrier ampholytes in combination. This so-called R2D2 buffer (5 M urea, 2 M thiourea, 2% 3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propane-sulfonate, 2% N-decyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate, 20 mM dithiothreitol, 5 mM Tris(2-carboxyethyl) phosphine, 0.5% carrier ampholytes 4-6.5, 0.25% carrier ampholytes 3-10) proved to be very efficient for a large range of different samples and allowed us to obtain two-dimensional gels of high resolution and quality.