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Water resource recovery facility (WRRF) modeling requires robust and reliable characterization of the wastewater to be treated. Poor characterization can lead to unreliable model predictions, which can have significant economic consequences when models are used to make important facility upgrade/expansion and operational decisions. Current wastewater characterization practice often involves a limited number of relatively short-duration intensive campaigns. On-going work at the Great Lakes Water Authority (GLWA) WRRF, serving 3.1 million residents in Southeast Michigan, provided an opportunity to conduct more detailed wastewater characterization over an annual cycle. The collection system includes a significant combined sewer component, and the WRRF provides primary and secondary treatment (high purity oxygen activated sludge) and phosphorus removal via ferric chloride addition. Detailed wastewater fractionation was conducted weekly over a one-year period. Daily conventional secondary influent and process operational data from that same period were used to evaluate the efficiency of various wastewater characterization strategies on the bioreactor mixed liquor volatile suspended solids (MLVSS) concentration calculated using an International Water Association (IWA) Activated Sludge Model Number 1 (ASM1) with minor modifications. An adaptive strategy consisting of a series of short-duration characterization campaigns, used to assess model fit for its intended purpose and continued until a robust and reliable model result, is recommended. Periods of unusual plant influent and/or operational conditions should be identified, and data from these periods potentially excluded from the analysis. Sufficient data should also be collected to identify periods when poor model structure, rather than wastewater characterization, leads to poor fit of the model to actual data.

TCP transcription factors are key regulators of angiosperm cell proliferation processes. It is unknown whether their regulatory growth capacities are conserved across land plants, which we examined in liverworts, one of the earliest diverging land plant lineages. We generated knockout mutants for MpTCP1, the single TCP-P clade gene in Marchantia polymorpha, and characterized its function by conducting cell proliferation and morphological analyses as well as messenger RNA expression, transcriptome, chemical, and DNA binding studies. Mptcp1ge lines show a reduced vegetative thallus growth and extra tissue formation in female reproductive structures. Additionally, mutant plants reveal increased hydrogen peroxide (H2 O2 ) levels and an enhanced pigmentation in the thallus caused by formation of secondary metabolites, such as aminochromes. MpTCP1 proteins interact redox dependently with DNA and regulate the expression of a comprehensive redox network, comprising enzymes involved in H2 O2 metabolism. MpTCP1 regulates Marchantia growth in a context-dependent manner. Redox sensitivity of the DNA binding capacity of MpTCP1 proteins provides a mechanism to respond to altered redox conditions. Our data suggest that MpTCP1 activity could thereby have contributed to diversification of land plant morphologies and to adaptations to abiotic and biotic challenges, as experienced by liverworts during early land plant colonization.

The Arabidopsis thaliana CC‐type glutaredoxin (GRX) ROXY1 and the bZIP TGA transcription factor (TF) PERIANTHIA (PAN) interact in the nucleus and together regulate petal development. The CC‐type GRXs exist exclusively in land plants, and in contrast to the ubiquitously occurring CPYC and CGFS GRX classes, only the CC‐type GRXs expanded strongly during land plant evolution. Phylogenetic analyses show that TGA TFs evolved before the CC‐type GRXs in charophycean algae. MpROXY1/2 and MpTGA were isolated from the liverwort Marchantia polymorpha to analyze regulatory ROXY/TGA interactions in a basal land plant. Homologous and heterologous protein interaction studies demonstrate that nuclear ROXY/TGA interactions are conserved since the occurrence of CC‐type GRXs in bryophytes and mediated by a conserved ROXY C‐terminus. Redox EMSA analyses show a redox‐sensitive binding of MpTGA to the cis‐regulatory as‐1‐like element. Furthermore, we demonstrate that MpTGA binds together with MpROXY1/2 to this motif under reducing conditions, whereas this interaction is not observed under oxidizing conditions. Remarkably, heterologous complementation studies reveal a strongly conserved land plant ROXY activity, suggesting an ancestral role for CC‐type GRXs in modulating the activities of TGA TFs. Super‐resolution microscopy experiments detected a strong colocalization of ROXY1 with the active form of the RNA polymerase II in the nucleus. Together, these data shed new light on the function of ROXYs and TGA TFs and the evolution of redox‐sensitive transcription regulation processes, which likely contributed to adapt land plants to novel terrestrial habitats.

Evolution of floral monosymmetry is thought to be a major driving force of angiosperm radiation, making angiosperms the most successful land plant group in terms of species richness. Monosymmetry evolved from a polysymmetric ancestor repeatedly in different angiosperm lineages, where it likely facilitated diversification through the interaction with insects. Most monosymmetric taxa are thus dominated by monosymmetric members. However, in the Brassicaceae, only few members develop a monosymmetric corolla with two petal pairs of unequal size, making them an ideal system to study the evolution of molecular mechanisms enhancing flower complexity. Monosymmetry is controlled by the TCP transcription factors that belong to the CYC2 clade in distantly related taxa. In Iberis amara, the first crucifer analyzed in terms of monosymmetry development, unequal corolla formation is due to a stronger CYC2 clade gene expression in the smaller adaxial petals compared with the larger abaxial ones. Phylogenetic reconstruction of the crucifer family reveals that the monosymmetric genera Iberis, Calepina, and Teesdalia belong to one major crucifer lineage. Monosymmetry is most pronounced in Iberis and less so in Calepina and Teesdalia, with a positive dosage-dependent correlation between the strength of a CYC2 expression difference and the extent of monosymmetry formation. An early adaxial CYC2 expression in floral meristems, observed in many distantly related taxa, might have facilitated the repeated evolution of CYC2-controlled monosymmetry. Comparison of early and late CYC2 expression in monosymmetric and polysymmetric crucifers representative for the four major crucifer lineages reveals that an adaxial CYC2 expression in floral meristems is likely ancestral for the Brassicaceae. However, it got lost in all analyzed monosymmetric members and is, as such, not a prerequisite for the establishment of corolla monosymmetry in crucifers. Here, monosymmetry evolved via a heterochronic CYC2 expression shift from an ancestral early adaxial expression in floral meristems to an adaxial CYC2 transcript accumulation later in petal development. This study emphasizes the potential of regulatory changes in the evolution of morphological novelties, like corolla monosymmetry in the Brassicaceae. In combination with a corymboid inflorescence, monosymmetry might have served as a key invention driving diversification in the genus Iberis comprising more than 20 monosymmetric species.

Background In the co-evolution between insects and plants, the establishment of floral monosymmetry was an important step in angiosperm development as it facilitated the interaction with insect pollinators and, by that, likely enhanced angiosperm diversification. In Antirrhinum majus, the TCP transcription factor CYCLOIDEA is the molecular key regulator driving the formation of floral monosymmetry. Although most Brassicaceae form a polysymmetric corolla, six genera develop monosymmetric flowers with two petal pairs of unequal size. In the monosymmetric crucifer Iberis amara, formation of the different petal pairs coincides with a stronger expression of the CYC-homolog IaTCP1 in the small, adaxial petals. Results In this study, RNA-Seq was employed to reconstruct the petal transcriptome of the non-model species Iberis amara. About 9 Gb of sequence data was generated, processed and re-assembled into 18,139 likely Iberis unigenes, from which 15,983 showed high sequence homology to Arabidopsis proteins. The transcriptome gives detailed insight into the molecular mechanisms governing late petal development. In addition, it was used as a scaffold to detect genes differentially expressed between the small, adaxial and the large, abaxial petals in order to understand the molecular mechanisms driving unequal petal growth. Far more genes are expressed in adaxial compared to abaxial petals implying that IaTCP1 activates more genes than it represses. Amongst all genes upregulated in adaxial petals, a significantly enhanced proportion is associated with cell wall modification and cell-cell signalling processes. Furthermore, microarrays were used to detect and compare quantitative differences in TCP target genes in transgenic Arabidopsis plants ectopically expressing different TCP transcription factors. Conclusions The increased occurrences of genes implicated in cell wall modification and signalling implies that unequal petal growth is achieved through an earlier stop of the cell proliferation phase in the small, adaxial petals, followed by the onset of cell expansion. This process, which forms the monosymmetric corolla of Iberis amara, is likely driven by the enhanced activity of IaTCP1 in adaxial petals. Electronic supplementary material The online version of this article (doi:10.1186/s12870-014-0285-4) contains supplementary material, which is available to authorized users.

Flower monosymmetry contributes to specialized interactions between plants and their insect pollinators. In the magnoliids, flower monosymmetry is exhibited only in the Aristolochiaceae (Piperales). Aristolochia flowers develop a calyx-derived monosymmetric perianth that enhances pollination success by a flytrap mechanism. Aristolochia arborea forms additionally a special perianth outgrowth that mimics a mushroom to attract flies, the mushroom mimicry structure (MMS). In core eudicots, members of the CYC2 clade of TCP transcription factors are key regulators of corolla monosymmetry establishment. The CYC2 clade arose via core eudicot-specific duplications from ancestral CYC/TB1 genes. CYC/TB1 genes are also thought to affect monosymmetry formation in early diverging eudicot and monocot species. Here, we demonstrate that CYC/TB1 genes, named CYC-like genes (CYCL) are present in basal angiosperms and magnoliids. Expression analyses in A. arborea indicate that CYCL genes participate in perianth and MMS differentiation processes and do not support a CYCL gene function in initial flower monosymmetry formation. Heterologous CYCL and CYC2 gene overexpression studies in Arabidopsis show that Aristolochia CYCL proteins only perform a CYC2-like function when the CYCL TCP domain is replaced by a CYC2 domain. Comparative TCP domain analyses revealed that an LxxLL motif, known to mediate protein-protein interactions, evolved in the second helix of the TCP domain in the CYC2 lineage and contributes to CYC2-related functions. Our data imply that divergent evolution of the CYC/TB1 lineages caused significant changes in their coding regions, which together with cis-regulatory changes established the key CYC2 function in regulating eudicot flower monosymmetry.

Glutaredoxins (GRXs) have thus far been associated mainly with redox-regulated processes participating in stress responses. However, ROXY1, encoding a GRX, has recently been shown to regulate petal primorida initiation and further petal morphogenesis in Arabidopsis thaliana. ROXY1 belongs to a land plant-specific class of GRXs that has a CC-type active site motif, which deviates from ubiquitously occurring CPYC and CGFS GRXs. Expression studies of yellow fluorescent protein-ROXY1 fusion genes driven by the cauliflower mosaic virus 35S promoter reveal a nucleocytoplasmic distribution of ROXY1. We demonstrate that nuclear localization of ROXY1 is indispensable and thus crucial for its activity in flower development. Yeast two-hybrid screens identified TGA transcription factors as interacting proteins, which was confirmed by bimolecular fluorescence complementation experiments showing their nuclear interaction in planta. Overlapping expression patterns of ROXY1 and TGA genes during flower development demonstrate that ROXY1/TGA protein interactions can occur in vivo and support their biological relevance in petal development. Deletion analysis of ROXY1 demonstrates the importance of the C terminus for its functionality and for mediating ROXY1/TGA protein interactions. Phenotypic analysis of the roxy1-2 pan double mutant and an engineered chimeric repressor mutant from PERIANTHIA (PAN), a floral TGA gene, supports a dual role of ROXY1 in petal development. Together, our results show that the ROXY1 protein functions in the nucleus, likely by modifying PAN posttranslationally and thereby regulating its activity in petal primordia initiation. Additionally, ROXY1 affects later petal morphogenesis, probably by modulating other TGA factors that might act redundantly during differentiation of second whorl organs.

Establishment of morphological novelties has contributed to the enormous diversification of floral architecture. One such novelty, flower monosymmetry, is assumed to have evolved several times independently during angiosperm evolution. To date, analysis of monosymmetry regulation has focused on species from taxa where monosymmetry prevails, such as the Lamiales and Fabaceae. In Antirrhinum majus , formation of a monosymmetric corolla is specified by the activity of the TCP transcription factors CYCLOIDEA ( CYC ) and DICHOTOMA ( DICH ). It was shown that establishment of monosymmetry likely requires an early asymmetric floral expression of CYC homologs that needs to be maintained until late floral stages. To understand how CYC homologs might have been recruited during evolution to establish monosymmetry, we characterized the likely CYC ortholog IaTCP1 from Iberis amara (Brassicaceae). Species of the genus Iberis form a monosymmetric corolla, whereas the Brassicaceae are otherwise dominated by genera developing a polysymmetric corolla. Instead of four equally sized petals, I. amara produces two small adaxial and two large abaxial petals. The timing of IaTCP1 expression differs from that of its Arabidopsis homolog TCP1 and other CYC homologs. IaTCP1 lacks an asymmetric early expression but displays a very strong differential expression in the corolla at later floral stages, when the strongest unequal petal growth occurs. Analysis of occasionally occurring peloric Iberis flower variants and comparative functional studies of TCP homologs in Arabidopsis demonstrate the importance of an altered temporal IaTCP1 expression within the Brassicaceae to govern the formation of a monosymmetric corolla.

Flower symmetry is considered a morphological novelty that contributed significantly to the rapid radiation of the angiosperms, which already puzzled Charles Darwin and prompted him to name this phenomenon an ‘abominable mystery’. In 2009, the bicentenary of Darwin's birth and the 150th anniversary of the publication of his seminal work, ‘On the Origin of Species’, this question can now be more satisfactorily readdressed. Understanding the molecular control of monosymmetry formation in the model species Antirrhinum opened the path for comparative studies with non-model species revealing modifications of this trait. TCP transcription factors, named after TEOSINTE BRANCHED 1 in maize, CYCLOIDEA in snapdragon and PCF in rice, control flower monosymmetry development and contributed to establishing this trait several times independently in higher angiosperms. The joint advances in evolutionary and developmental plant research, combined in the novel research field named Evo/Devo, aim at elucidating the molecular mechanisms and strategies to unravel the mystery of how this diversity has been generated.