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Mixed cultures of Sertoli and germ cells were prepared from rat testes and their response to lead (Pb) was studied. Cultures consisted of a monolayer of Sertoli cells to which clusters of germ cells were attached. The effect of Pb added as lead acetate was tested at 0.0, 0.4, 4.0 and 40.0 microM for 24 and 48 h intervals. Addition of Pb to the culture medium caused germ cells to progressively detach from the Sertoli cell monolayer into the medium in a concentration and duration dependent manner Viability of the detached cells as judged by trypan blue exclusion test showed a decrease with increase in time and concentration of Pb. Significant leakage of lactate dehydrogenase (LDH) was recorded in the culture media only at the higher concentrations of 4.0 and 40.0 microM. Thus Pb at the doses tested induced cytotoxicity in rat Sertoli-germ cell coculture.

Endosulfan induced testicular impairment has been reported in vivo in rats. The present study was conducted to evaluate the cytotoxic effects of endosulfan in vitro employing rat testicular cells in culture, that is Sertoli–germ cell coculture. Cytotoxic changes induced by endosulfan (0, 2, 20, 40, and 80 μM) in mixed cultures of Sertoli and germ cells were seen after 24 and 48 h of treatment. Endosulfan led to an increase in germ cell detachment from the Sertoli cell monolayer in a dose dependent manner. A loss in the viability of detached cells was observed in all treated groups. Substantial leakage of the cytosolic enzyme lactate dehydrogenase was observed in the medium after 24 and 48 h endosulfan treatment at concentrations of 20 to 80 μM. The extent of toxicity was greater after 48 h of treatment.

The plant epidermis is only rarely involved in fusion events. These events often occur when the epidermis has been wounded or removed. The adherentl (adl) mutation in maize shows fusions between cells and organs in the presence of an intact epidermis. Fusions are not limited to a particular stage of organ development. The first fusion events occur very early in development and adherent leaves can be seen in ungerminated embryos. In the vegetative parts of the plant the coleoptile can fuse to the first leaf and juvenile leaves can fuse to each other. At the onset of reproduction, tassel branches can adhere to each other, the flag leaf can fuse to the tassel, and the glumes can fuse to the anthers. While fused regions appear to have an intact cuticle, cell surfaces are perturbed in the mutation. Time-lapse replicas reveal that trichome fusion on organ surfaces is postgenital. The extracellular matrix can exert control over organs and tissues in plants; the Adl gene may represent a product that determines th...

In the absence of wounding, the epidermis is only rarely involved in cell or organ fusion events; in fact, intact epidermal layers prevent graft unions. In Zea mays L. the mutation adherent1 (ad1) shows abnormal fusions between cells and organs. Fusions involve epidermal cells of vegetative and floral organs and occur early in the ontogeny of organs. Even so, epidermal cell types differentiate normally in the fused regions and internal tissue identities are maintained. In contrast, the extracellular matrix (cell wall and cuticle) of the epidermal cells is perturbed. Epidermal cell walls in adherent leaves are thicker than normal. Epicuticular wax particles appear reduced in size and number and altered in shape in mutant leaves. In addition, the outer epidermal cell walls of adherent leaves fluoresce when stained with aniline blue, a reagent that binds to callose. Immunolocalizations to specific cell wall epitopes suggest that pectins but not arabinogalactans may have a role in the fusion events. Taken together, these results suggest that the ad1 mutation results in cell-wall and epicuticular-wax defects similar to responses seen in wounding, pollination by incompatible pollen, or pathogen attack. Since cell wall components and epicuticular waxes are extracellular secreted products, the ad1 mutation may disrupt normal functioning and/or composition of the secretory pathway and its cargo.

The cultivated tomato (Lycopersicon esculentum) has a unipinnate compound leaf. In the developing leaf primordium, major leaflet initiation is basipetal, and lobe formation and early vascular differentiation are acropetal. We show that engineered alterations in the expression of a tomato homeobox gene, LeT6, can cause dramatic changes in leaf morphology. The morphological states are variable and unstable and the phenotypes produced indicate that the tomato leaf has an inherent level of indeterminacy. This is manifested by the production of multiple orders of compounding in the leaf, by numerous shoot, inflorescence, and floral meristems on leaves, and by the conversion of rachis-petiolule junctions into “axillary” positions where floral buds can arise. Overexpression of a heterologous homeobox transgene,kn1, does not produce such phenotypic variability. This indicates that LeT6 may differ from the heterologouskn1 gene in the effects manifested on overexpression, and that 35S-LeT6 plants may be subject to alterations in expression of both the introduced and endogenous LeT6genes. The expression patterns of LeT6 argue in favor of a fundamental role for LeT6 in morphogenesis of leaves in tomato and also suggest that variability in homeobox gene expression may account for some of the diversity in leaf form seen in nature.

Homeobox genes are known to play a role in developmental regulation. The knotted-like homeobox (knox) genes fall into two classes. The class I knox genes like kn1, stm1, and knat1 are involved in maintaining meristem identity in cells. The function of class II knox genes is at yet undetermined. We have characterized two knox genes from tomato. LeT6 and LeT12 map to distinct chromosome locations that are different from the location for a recently cloned knox gene from tomato, tkn1, confirming that plant homeobox genes are not clustered on chromosomes. These genes have a distinct expression pattern. Unlike other class I kn1-like genes, LeT6 is expressed in developing lateral organs and developing ovaries in flowers. LeT12 is more ubiquitously expressed in the mature plant. RNA in situ localization data suggest that both these genes may have a role to play in formative events in ovule and embryo morphogenesis.

Björn Usadel and colleagues report the genome sequence of the wild tomato species Solanum pennellii. The authors identify genes important for stress tolerance, metabolism and fruit maturation and suggest that transposable elements have had an important role in the evolution of the S. penellii stress response. Supplementary information The online version of this article (doi:10.1038/ng.3046) contains supplementary material, which is available to authorized users., Solanum pennellii is a wild tomato species endemic to Andean regions in South America, where it has evolved to thrive in arid habitats. Because of its extreme stress tolerance and unusual morphology, it is an important donor of germplasm for the cultivated tomato Solanum lycopersicum1. Introgression lines (ILs) in which large genomic regions of S. lycopersicum are replaced with the corresponding segments from S. pennellii can show remarkably superior agronomic performance2. Here we describe a high-quality genome assembly of the parents of the IL population. By anchoring the S. pennellii genome to the genetic map, we define candidate genes for stress tolerance and provide evidence that transposable elements had a role in the evolution of these traits. Our work paves a path toward further tomato improvement and for deciphering the mechanisms underlying the myriad other agronomic traits that can be improved with S. pennellii germplasm. Supplementary information The online version of this article (doi:10.1038/ng.3046) contains supplementary material, which is available to authorized users.

Homeodomain proteins are transcription factors that play a critical role in early development in eukaryotes. These proteins previously have been classified into numerous subgroups whose phylogenetic relationships are unclear. Our phylogenetic analysis of representative eukaryotic sequences suggests that there are two major groups of homeodomain proteins, each containing sequences from angiosperms, metazoa, and fungi. This result, based on parsimony and neighbor-joining analyses of primary amino acid sequences, was supported by two additional features of the proteins. The two protein groups are distinguished by an insertion/deletion in the homeodomain, between helices I and II. In addition, an amphipathic alpha-helical secondary structure in the region N terminal of the homeodomain is shared by angiosperm and metazoan sequences in one group. These results support the hypothesis that there was at least one duplication of homeobox genes before the origin of angiosperms, fungi, and metazoa. This duplication, in turn, suggests that these proteins had diverse functions early in the evolution of eukaryotes. The shared secondary structure in angiosperm and metazoan sequences points to an ancient conserved functional domain.

Flowering plants can have either simple or compound leaves. Compound leaves have an interesting evolutionary history, and arose independently in the ferns, cycads and flowering plants. The compound structure may be a consequence of simple leaves that are subdivided into separate leaflets; inherently different from simple leaves, and intermediate between shoots and leaves; or the leaf ground state, with suppression and reduction leading to simple leaves. The knox genes have been extensively studied to determine their role in leaf initiation and development. Preliminary evidence indicates that the simple and compound leaf types, though superficially similar at the time of initiation, have differences that arise in the preprimordium stage in the shoot apical meristem. Compound leaves in peas and tomato probably have independent origins from an ancestrally simple state in the basal angiosperms. Genetic and molecular analyses of leaf morphogenesis in diverse organisms will help determine whether independent origins of compound leaves gave rise to similar structures.

TCP family of plant-specific transcription factors regulates plant form through control of cell proliferation and differentiation. This gene family is comprised of two groups, class I and class II. While the role of class II TCP genes in plant development is well known, data about the function of some class I TCP genes is lacking. We studied a group of phylogenetically related class I TCP genes: AtTCP7, AtTCP8, AtTCP22, and AtTCP23. The similar expression pattern in young growing leaves found for this group suggests similarity in gene function. Gene redundancy is characteristic in this group, as also seen in the class II TCP genes. We generated a pentuple mutant tcp8 tcp15 tcp21 tcp22 tcp23 and show that loss of function of these genes results in changes in leaf developmental traits. We also determined that these factors are able to mutually interact in a yeast two-hybrid assay and regulate the expression of KNOX1 genes. To circumvent the issue of genetic redundancy, dominant negative forms with SRDX repressor domain were used. Analysis of transgenic plants expressing AtTCP7-SRDX and AtTCP23-SRDX indicate a role of these factors in the control of cell proliferation.

The variability in leaf morphology is vast; therefore, an understanding of how leaves achieve this diversity has captured the attention of many developmental biologists. Genetic mechanisms that regulate leaf shape are repeated throughout both development and between species. Leaves initiate off the shoot apical meristem and development proceeds from cell division to cell elongation. Leaf initiation and the establishment of the three main axes of a leaf are largely directed by the plant hormone auxin. Work across species reveals that adjustments to the repeated interaction of auxin, gene expression changes and small ribonucleic acid (RNA) regulation allowed species-specific leaf shape differences to emerge. Further, genetic interactions leading to patterning in simple leaf development are reiterated during establishment of leaf complexity. To understand leaf development in an evolutionary context, future research must take advantage of the ever-expanding genomic tools in nonmodel organisms that exhibit leaf shape diversity. Key Concepts: Leaves originate from clusters of stem cells called meristems. The development of leaves occurs along three main axes: (1) adaxial/abaxial, (2) proximal/distal and (3) medial/lateral. The development of both meristems and leaves involve morphogenetic activity in zones which are defined by distinct gene expression and cell division/elongation patterns. The transport and concentration of the hormone auxin largely directs many aspects of leaf morphogenesis. Leaf and leaflet initiation first begin with the formation of auxin maxima. Recycling of genetic mechanisms is a theme that occurs throughout leaf development. Keywords: abaxial; adaxial; HDZIPIII ; KANADI ; leaf; marginal blastozone; miRNA; polarity; PIN-FORMED ; YABBY

Plant secondary cell walls are deposited mostly in vascular tissues such as xylem vessels, tracheids, and fibers. These cell walls are composed of a complex matrix of compounds including cellulose, hemicellulose, and lignin. Lignin functions primarily to maintain the structural and mechanical integrity of both the transport vessel and the entire plant itself. Since lignin has been identified as a major source of biomass for biofuels, regulation of secondary cell wall biosynthesis has been a topic of much recent investigation. Biosynthesis and patterning of lignin involves many developmental and environmental cues including evolutionarily conserved transcriptional regulatory modules and hormonal signals. Here, we investigate the role of the class I Knotted1-like-homeobox (KNOX) genes and gibberellic acid in the lignin biosynthetic pathway in a representative monocot and a representative eudicot. Knotted1 overexpressing mutant plants showed a reduction in lignin content in both maize and tobacco. Expression of four key lignin biosynthesis genes was analyzed and revealed that KNOX1 genes regulate at least two steps in the lignin biosynthesis pathway. The negative regulation of lignin both in a monocot and a eudicot by the maize Kn1 gene suggests that lignin biosynthesis may be preserved across large phylogenetic distances. The evolutionary implications of regulation of lignification across divergent species are discussed.

C4 photosynthesis is thought to be an adaptation to warm environments, involving complex changes in the expression of genes governing photosynthesis, intermediary metabolism, and leaf anatomy and histology. Such complexity should be difficult to evolve, yet the pathway has arisen multiple times in the history of the flowering plants and at least four times in the grass family alone. We have used immunolocalization techniques to compare photosynthetic gene expression across all four origins, to determine which genetic changes occur in parallel and which are unique to a particular lineage. The only gene expression patterns common to all origins of the pathway are up-regulation of PEP carboxylase and down-regulation of RuBisCO in mesophyll cells. Both NAD-malic enzyme and NADP-malic enzyme are expressed in bundle sheaths. Expression patterns of light-harvesting chlorophyll a/b binding proteins and pyruvate orthophosphate dikinase appear to be lineage specific, and may be localized to bundle sheaths or to mesophyll or expressed throughout the photosynthetic tissue of the leaf. We suggest that future studies of parallel origin of the C4 pathway concentrate on regulation of the two carboxylases, as well as the increased density of vascular tissue, which is the only histological characteristic common to all origins of the pathway.

Leaf morphology and the pattern of shoot branching determine to a large extent the growth habit of seed plants. Until recently, the developmental processes that led to the establishment of these morphological structures seemed unrelated. Here, we show that the tomato Trifoliate ( Tf ) gene plays a crucial role in both processes, affecting the formation of leaflets in the compound tomato leaf and the initiation of axillary meristems in the leaf axil. Tf encodes a myeloblastosis oncoprotein (MYB)-like transcription factor related to the Arabidopsis thaliana LATERAL ORGAN FUSION1 (LOF1) and LOF2 proteins. Tf is expressed in the leaf margin, where leaflets are formed, and in the leaf axil, where axillary meristems initiate. During tomato ontogeny, expression of Tf in young leaf primordia increases, correlating with a rise in leaf dissection (heteroblasty). Formation of leaflets and initiation of axillary meristems can be traced back to groups of pluripotent cells. Tf function is required to inhibit differentiation of these cells and thereby to maintain their morphogenetic competence, a fundamental process in plant development. KNOTTED1-LIKE proteins, which are known regulators in tomato leaf dissection, require Tf activity to exert their function in the basal part of the leaf. Similarly, the plant hormone auxin needs Tf activity to initiate the formation of lateral leaflets. Thus, leaf dissection and shoot branching rely on a conserved mechanism that regulates the morphogenetic competence of cells at the leaf margin and in the leaf axil.

International audience; Although applied over extremely short timescales, artificial selection has dramatically altered the form, physiology, and life history of cultivated plants. We have used RNAseq to define both gene sequence and expression divergence between cultivated tomato and five related wild species. Based on sequence differences, we detect footprints of positive selection in over 50 genes. We also document thousands of shifts in gene-expression level, many of which resulted from changes in selection pressure. These rapidly evolving genes are commonly associated with environmental response and stress tolerance. The importance of environmental inputs during evolution of gene expression is further highlighted by large-scale alteration of the light response coexpression network between wild and cultivated accessions. Human manipulation of the genome has heavily impacted the tomato transcriptome through directed admixture and by indirectly favoring nonsynonymous over synonymous substitutions. Taken together, our results shed light on the pervasive effects artificial and natural selection have had on the transcriptomes of tomato and its wild relatives.

International audience; Introgression lines (ILs), in which genetic material from wild tomato species is introgressed into a domesticated background, have been used extensively in tomato (Solanum lycopersicum) improvement. Here, we genotype an IL population derived from the wild desert tomato Solanum pennellii at ultrahigh density, providing the exact gene content harbored by each line. To take advantage of this information, we determine IL phenotypes for a suite of vegetative traits, ranging from leaf complexity, shape, and size to cellular traits, such as stomatal density and epidermal cell phenotypes. Elliptical Fourier descriptors on leaflet outlines provide a global analysis of highly heritable, intricate aspects of leaf morphology. We also demonstrate constraints between leaflet size and leaf complexity, pavement cell size, and stomatal density and show independent segregation of traits previously assumed to be genetically coregulated. Meta-analysis of previously measured traits in the ILs shows an unexpected relationship between leaf morphology and fruit sugar levels, which RNA-Seq data suggest may be attributable to genetically coregulated changes in fruit morphology or the impact of leaf shape on photosynthesis. Together, our results both improve upon the utility of an important genetic resource and attest to a complex, genetic basis for differences in leaf morphology between natural populations.

The genome sequence of tomato (Solanum lycopersicum), one of the most important vegetable crops, has recently been decoded. We address implications of the tomato genome for plant breeding, genomics and evolutionary studies, and its potential to fuel future crop biology research.

Spiral phyllotactic patterning is the result of intricate auxin transport relationships in the shoot apical meristem (SAM) that act to place auxin maxima at the future sites of leaf initiation. Inherent to this process is a bias in auxin distribution in leaf primordia, such that increased auxin is found on the descending side of the leaf (towards the older neighbor) compared to the ascending side (towards the younger neighbor), creating phyllotactically-dependent leaf asymmetry. Separate from phyllotactic-dependent asymmetry is handedness in plants—that is, genetically encoded, fixed chirality, such as the twining of certain vines and the torsions induced by microtubule mutations. Here, we perform a morphometric analysis on the resupinate leaves of Alstroemeria psittacina. Interestingly, the twist in leaves always occurs in a single direction, regardless of the phyllotactic direction of the plant. Because of the resupination, leaves in this species possess an inherent handedness. However, this asymmetry is modulated in a phyllotactic-dependent manner, consistent with the known developmental constraints of phyllotaxis upon leaf morphology. This creates the interesting circumstance in A. psittacina that leaves arising from plants with a counter-clockwise phyllotactic direction are 1) more asymmetric, 2) larger, and 3) possess symmetrical shape differences relative to leaves from plants with clockwise phyllotaxis. The mechanism underlying these differences likely involves a developmental delay in clockwise leaves caused by the conflict between the phyllotaxis-dependent asymmetry and asymmetry resulting from resupination. The evolutionary implications of a dimorphic population without a genetic basis for selection to act upon are discussed.

Background Striga species are noxious root hemi-parasitic weeds that debilitate cereal production in sub-Saharan Africa (SSA). Control options for Striga are limited and developing Striga resistant crop germplasm is regarded as the best and most sustainable control measure. Efforts to improve germplasm for Striga resistance by a non-Genetic Modification (GM) approach, for example by exploiting natural resistance, or by a GM approach are constrained by limited information on the biological processes underpinning host-parasite associations. Additionaly, a GM approach is stymied by lack of availability of candidate resistance genes for introduction into hosts and robust transformation methods to validate gene functions. Indeed, a majority of Striga hosts, the world’s most cultivated cereals, are recalcitrant to genetic transformation. In maize, the existing protocols for transformation and regeneration are tedious, lengthy, and highly genotype-specific with low efficiency of transformation. Results We used Agrobacterium rhizogenes strain K599 carrying a reporter gene construct, Green Fluorescent Protein (GFP), to generate transgenic composite maize plants that were challenged with the parasitic plant Striga hermonthica. Eighty five percent of maize plants produced transgenic hairy roots expressing GFP. Consistent with most hairy roots produced in other species, transformed maize roots exhibited a hairy root phenotype, the hallmark of A. rhizogenes mediated transformation. Transgenic hairy roots resulting from A. rhizogenes transformation were readily infected by S. hermonthica. There were no significant differences in the number and size of S. hermonthica individuals recovered from either transgenic or wild type roots. Conclusions This rapid, high throughput, transformation technique will advance our understanding of gene function in parasitic plant-host interactions.

The laminae of leaves optimize photosynthetic rates by serving as a platform for both light capture and gas exchange, while minimizing water losses associated with thermoregulation and transpiration. Many have speculated that plants maximize photosynthetic output and minimize associated costs through leaf size, complexity, and shape, but a unifying theory linking the plethora of observed leaf forms with the environment remains elusive. Additionally, the leaf itself is a plastic structure, responsive to its surroundings, further complicating the relationship. Despite extensive knowledge of the genetic mechanisms underlying angiosperm leaf development, little is known about how phenotypic plasticity and selective pressures converge to create the diversity of leaf shapes and sizes across lineages. Here, we use wild tomato accessions, collected from locales with diverse levels of foliar shade, temperature, and precipitation, as a model to assay the extent of shade avoidance in leaf traits and the degree to which these leaf traits correlate with environmental factors. We find that leaf size is correlated with measures of foliar shade across the wild tomato species sampled and that leaf size and serration correlate in a species-dependent fashion with temperature and precipitation. We use far-red induced changes in leaf length as a proxy measure of the shade avoidance response, and find that shade avoidance in leaves negatively correlates with the level of foliar shade recorded at the point of origin of an accession. The direction and magnitude of these correlations varies across the leaf series, suggesting that heterochronic and/or ontogenic programs are a mechanism by which selective pressures can alter leaf size and form. This study highlights the value of wild tomato accessions for studies of both morphological and light-regulated development of compound leaves, and promises to be useful in the future identification of genes regulating potentially adaptive plastic leaf traits.

With the introduction of cost effective, rapid and superior quality next generation sequencing (NGS) techniques, gene expression analysis has become viable for labs conducting small projects as well as large-scale gene expression analysis experiments. However, the available protocols for construction of RNA-Sequencing (RNA-Seq) libraries are expensive and/or difficult to scale for high-throughput applications. Also, most protocols require isolated total RNA as a starting point. We provide a cost-effective RNA-Seq library synthesis protocol that is fast, starts with tissue, and is high-throughput from tissue to synthesized library. We have also designed and report a set of 96 unique barcodes for library adapters that are amenable to high-throughput sequencing by a large combination of multiplexing strategies. Our developed protocol has more power to detect differentially expressed genes when compared to the standard Illumina protocol, probably owing to less technical variation amongst replicates. We also address the problem of gene-length biases affecting differential gene expression calls and demonstrate that such biases can be efficiently minimized during mRNA isolation for library preparation.

The dominant Knotted-1 mutations in maize alter development of the leaf blade. Sporadic patches of localized growth, or knots, and fringes of ectopic ligule occur along lateral veins of mutant leaf blades. In addition, bundle sheaths do not completely encircle lateral veins on mutant leaf blades. We have compared mutant leaf blades with wild-type leaves to determine the precise nature of the perturbed regions. Our analysis includes characterization of epidermal cell shapes, localization of photosynthetic proteins and histology of the leaf. We show that mutant leaf blades are a mosaic of leaf organ components. Affected regions of mutant leaf blades resemble either sheath or auricle tissue in both external and internal features. This conversion of blade cells represents an acropetal shift of more basal parts of the leaf blade region and correlates with previously identified ectopic expression of the Knotted-1 protein in the leaf blade. We propose that inappropriate expression of Kn1 interferes with the process of establishment of cell identities, resulting in early termination of the normal blade development program or precocious expression of the sheath and auricle development programs. © 1994 Wiley-Liss, Inc.

KNOTTEDI-like homeobox (KNOXI) genes regulate development of the leaf from the shoot apical meristem (SAM) and may regulate leaf form. We examined KNOXI expression in SAMs of various vascular plants and found that KNOXI expression correlated with complex leaf primordia. However, complex primordia may mature into simple leaves. Therefore, not all simple leaves develop similarly, and final leaf morphology may not be an adequate predictor of homology.

At around the same time in the late 1960s, two great minds grappled with two seemingly different questions. Lewis Wolpert tackled the problem of how specific identities can be assigned to seemingly identical cells to generate a pattern. Theodosius Dobzhansky wondered why the living world was NOT a mass of one kind of living things, surviving in one special environment. Wolpert went on to use Turing's ideas of how pattern can be generated in a uniform field to provide positional information, which is then utilized to generate molecular readouts (Wolpert, 1969). Dobzhansky, in turn, described how combinations of genes could form a large but finite number of adaptive peaks, and each species then occupied a particular adaptive peak with reference to the gene combinations it utilized. The generation of heterogeneity in a sea of sameness was achieved at the levels of cells over time in development and at the level of individuals in a species during the course of evolution (Dobzhansky, 1970). A few years later, the two fields underwent a friendly collision, most notably with the publication of Gould's Ontogeny and Phylogeny, the attempt to relate embryonic development to evolution (Gould, 1977). Developmental Biology itself progressed from classic descriptive and experimental studies on the generation of form, to the study of genes, and underlying genetic mechanisms. Once genes were identified one could look at the commonality or differences in developmental mechanisms across various phylogenetic levels. The use of reporter genes and in situ expression analyses filled in the details on where a gene product was present and the study of genetic mutations allowed elucidation of gene function. The gene-by-gene approach proved very fruitful and, combined with genetic analyses, allowed for the elucidation of developmental pathways in several model species. The focus on Arabidopsis thaliana as the premier model system, with the first completely sequenced genome, pushed the frontiers of plant developmental biology in the last decade. However, the advent of genomics promised to change all fields of biological inquiry and make any organism accessible for study. There is no doubt having a sequenced genome makes an organism instantly amenable to many kinds of analyses. Chromosome walking, an exercise that consumed the entire career of a graduate student in the past, is now more like chromosome gazing for gene identification. One has instant access to promoters, gene structure, and, in some organisms, access to genome variation across accessions or cultivars. The ILLUMINA sequencing platform can now provide transcriptome profile data on a genome scale. When used between accessions, transcriptome profiling can, in addition to registering subtle changes in gene expression levels, provide information on variation at the level of sequence. Other emerging technologies have been coupled with genomics to generate very precise expression catalogs for tissues and cell types. These technologies include sorting of marked cells or tissues (Birnbaum et al., 2003), or laser capture microdissection (LCM) of cells or tissues (Kerk et al., 2003), followed by transcriptome profiling. The challenges after such profiles are generated depend on how tractable the organism is for laboratory research and the questions that it is uniquely suited to answering. Research approaches can vary vastly between the well-developed models and non-models when it comes to understanding developmental mechanisms based on expression profiling. Comparison of global expression profiles of various floral organs across the Angiosperm phylogeny provided insights into the origin and subsequent diversification of flowers (Chanderbali et al., 2010). While functional validation of hypotheses poses a challenge in non-model systems, in the well-developed models the power of reverse genetics can prove very fruitful. Recent development of virus induced gene silencing (VIGS) in dicot (Dinesh-Kumar et al., 2003) and monocot plants (Holzberg et al., 2002; Scofield and Nelson, 2009) has brought reverse genetics, though in a transient and not stable mode, to many non-model species (Di Stilio et al., 2010; Preston and Hileman, 2010). The clout provided by affordable and accessible high-throughput sequencing (along with limited reverse genetics tools) now opens up many classic questions to fresh ways of inquiry. However, while the selection of organisms to be sequenced has certain drivers – phylogenetic placement, unique morphologies, curious ecological niches, economic importance – none of these aims match with what makes an organism a suitable developmental model. At the very least factors like ease of cultivation under laboratory conditions, and the ability to take the organism through its complete life cycle within a reasonable time span, have to be considered when developing new model organisms. Some issues we need to consider when it comes to future efforts to improve our knowledge of plant growth and development include: 1 Careful choice of organisms for genome initiatives. Considerations should include, in addition to the obvious factors like genome size and ploidy level, the viability as a genetic model system, and ease of experimental manipulation. 2 Transgenic technology needs to be streamlined and developed for all major plant clades. VIGS is an alternative but not well-developed for all clades, shows incomplete silencing, and study of genetic interactions is virtually impossible on the VIGS platform. 3 The most important need of the hour is high-throughput phenotyping to match the high-throughput genotyping efforts going on. 4 Above all, we have a crisis in training – a lack of researchers who are trained to analyze and present data quantitatively, be it sequences, or high-throughput phenotyping, or modeling of developmental data. Of particular importance, in the world of changing ecological and climatological niches, will be our ability to identify organisms that inform on how plants can grow, develop, and adapt to changing conditions. The facility with which we can modify certain plants to meet our needs of food, feed, and fuel is tied into our understanding of how these organisms respond to their environments as they grow and develop. While growth measurements at the crude level can be automated, microscopic measurements are still a challenge, and a critical part of developmental biology – the ability to view development in time has only been achieved in very few systems (Reddy et al., 2004, 2007). The ability to model plant growth based on empirical data is growing by leaps and bounds (Jonsson and Krupinski, 2010; Prusinkiewicz and Barbier De Reuille, 2010), the next challenge is to utilize these models to predict genetic or developmental outcomes when key parameters are altered or key factors are missing, and then to test these models in systems that are genetically perturbed. Plant Developmental Biology is on the threshold of wonderful discoveries as we begin to capitalize on genomics, evolutionary biology, and the efforts to decipher the relationships between all living things in the Tree of Life. The future looks promising as new quantitative and computer aided modeling tools, and precision dissection of gene expression, are being combined with high-throughput sequencing to answer age old questions of signaling and cell fate commitment during development of an organism.

INTRODUCTIONTransformation is an essential technique to analyze the function of genes. Tomato can be stably transformed using Agrobacterium tumefaciens-mediated transfer of T-DNA. This protocol describes an Agrobacterium-mediated transformation method for tomato.

INTRODUCTIONTomato (Solanum lycopersicum) is one of the most important vegetable plants in the world. It originated in western South America, and domestication is thought to have occurred in Central America. Because of its importance as food, tomato has been bred to improve productivity, fruit quality, and resistance to biotic and abiotic stresses. Tomato has been widely used not only as food, but also as research material. The tomato plant has many interesting features such as fleshy fruit, a sympodial shoot, and compound leaves, which other model plants (e.g., rice and Arabidopsis) do not have. Most of these traits are agronomically important and cannot be studied using other model plant systems. There are 13 recognized wild tomato species that display a great variety of phenotypes and can be crossed with the cultivated tomato. These wild tomatoes are important for breeding, as sources of desirable traits, and for evolutionary studies. Current progress on the tomato genome sequencing project has generated useful information to help in the study of tomato. In addition, the tomato belongs to the extremely large family Solanaceae and is closely related to many commercially important plants such as potato, eggplant, peppers, tobacco, and petunias. Knowledge obtained from studies conducted on tomato can be easily applied to these plants, which makes tomato important research material. Because of these facts, tomato serves as a model organism for the family Solanaceae and, specifically, for fleshy-fruited plants.

INTRODUCTIONThis protocol describes how to cross tomato plants. Crossing is important for the genetic analysis and breeding of tomatoes. Tomatoes are self-pollinating plants; thus, emasculation (removal of the anthers from the female parent) is essential. All wild tomato species can be crossed with cultivated tomatoes (although it may be difficult); this is useful because wild tomatoes are a great source of desirable traits. Most commercial tomatoes are F1 hybrids, and the seeds for them were produced by crossing two parent tomatoes.

Biological crop pests cause serious economic losses. In Africa, the most prevalent parasites are insect pests, plant pathogenic root-knot nematodes, viruses and parasitic plants. African smallholder farmers struggle to overcome these parasitic constraints to agricultural production. Crop losses and the host range of these parasites have continued to increase in spite of the use of widely advocated control methods. A sustainable method to overcome biological pests in Africa would be to develop crop germplasm resistant to parasites. This is achievable using either genetic modification (GM) or a non-GM approach. However, there is a paucity of resistant genes available for introduction. Additionally, the biological processes underpinning host parasite resistance are not sufficiently well understood. The authors review a technology platform for using RNA-mediated interference (RNAi) as bioengineered resistance to important crop parasites in Africa. To achieve acquired resistance, a host crop is stably transformed with a transgene that encodes a hairpin RNA targeting essential parasitic genes. The RNAi sequence is chosen in such a way that it shares no homology with the host's genes, so it remains 'inactive' until parasitism. Upon parasitism, the RNAi sequence enters the parasite and post-transcriptional gene silencing (PTGS) mechanisms are activated, leading to the death of the parasite.

Leaf shape is a highly variable trait. Ancestrally, all leaves are proposed to have derived from modifications of branched shoot systems. The formation of blade, smooth margins or serrations on the blade, or distinct leaflets that are the characteristic features of some leaves provides an opportunity to study the generation of morphogenesis in organs that are evolutionarily homologous and yet developmentally distinct in patterning. Intense research in several model species with distinct leaf morphologies has revealed a complex network of genes that interact to pattern the leaf. Several parallels between leaf patterning and shoot patterning exist. The plant growth hormone auxin is emerging as a key player in the specification of both shoot and leaf patterning. The outcome of this underlying auxin pattern may be determined by variation in the two opposing developmental forces of differentiation and indeterminancy. The expanded suite of genetic and physiological factors regulating leaf shape has provided interesting insight into the mechanisms by which morphological innovation is accomplished.

S-allyl/propargyl/methallyl thioimminium salts derived from methyl 1-benzyl-5-thioxoprolinate ( 1 ) undergoes a facile thio-Claisen rearrangement which provides an easy access to 4-substituted thioxoprolinates ( 2–4 ).

Plant shoot organs arise from initial cells that are recruited from meristematic tissues. Previous studies have shown that members of the WUSCHEL-related HOMEOBOX (WOX) gene family function to organize various initial cell populations during plant development. The function of the WOX4 gene is previously undescribed in any plant species. Comparative analyses of WOX4 transcription and function are presented in Arabidopsis (Arabidopsis thaliana), a simple-leafed plant with collateral vasculature, and in tomato (Solanum lycopersicum), a dissected-leafed species with bicollateral venation. WOX4 is transcribed in the developing vascular bundles of root and shoot lateral organs in both Arabidopsis and tomato. RNA interference-induced down-regulation of WOX4 in Arabidopsis generated small plants whose vascular bundles accumulated undifferentiated ground tissue and exhibited severe reductions in differentiated xylem and phloem. In situ hybridization analyses of Atwox4-RNA interference plants revealed delayed and reduced expression of both the phloem developmental marker ALTERED PHLOEM1 and HOMEOBOX GENE8, a marker of the vascular procambium. Overexpression of SlWOX4 correlated with overproliferation of xylem and phloem in transgenic tomato seedlings. The cumulative data suggest that the conserved WOX4 function is to promote differentiation and/or maintenance of the vascular procambium, the initial cells of the developing vasculature.

INTRODUCTIONThe genus Kalanchoë plays an important role in the investigation of biochemical, physiological and phylogenetic aspects of Crassulacean acid metabolism (CAM) in plants, which is an important evolutionary adaptation of the photosynthetic carbon assimilation pathway to arid environments. In addition, natural compounds extracted from tissues of Kalanchoë have potential applicability in treating tumors and inflammatory and allergic diseases, and have been shown to have insecticidal properties. Kalanchoë daigremontiana (Hamet & Perrier) originated in Madagascar and reproduces asexually by spontaneously forming whole plantlets on leaves. Plantlets develop symmetrically along the leaf margins on leaf notches, closely resembling zygotic embryos in development, and once the root system is formed, they detach from the mother-leaf, fall to the ground, and grow into new plants. This phenomenon is also found in other species from this same genus; however, the formation of leaf-plantlets is variable among species. Nevertheless, all species illustrate the remarkable ability of plant somatic cells to regenerate an entire organism, which has fascinated the scientific community for many years. It was only recently that the morphogenic process involved in the origin of K. daigremontiana plantlets was determined using molecular and genetic tools: K. daigremontiana forms plantlets by co-opting both organogenesis and embryogenesis programs into leaves. The ability of K. daigremontiana species to form somatic embryos outside of a seed environment provides an attractive model system to study somatic embryogenesis in nature, particularly the molecular mechanism involved in the acquisition of competence by vegetative cells to make embryos without fertilization.

INTRODUCTIONThis protocol describes how to isolate total RNA from several tissues of Kalanchoë daigremontiana. Total RNA can be isolated by using the TRI-reagent method, scaled up for processing 2 g of tissue, or by using the protocol described here, which gives higher concentrations of high quality RNA. The resulting RNA can be used for various applications including generation of cDNA for reverse transcriptase-PCR (RT-PCR), Northern blots, or other purposes.

The profiles of thyroxine (T4) and triiodothyronine (T3) in serum were monitored at 4 h intervals over a 24 h cycle in nine months of the year in females of the freshwater catfish. Clarias batrachus by means of radioimmunoassay. The results exhibited significant seasonal changes of the daily cycles in the titres of T4 and T3. Interestingly, unlike earlier reports, T4 and T3 showed two peaks in the serum; one in the photophase and the other in the scotophase of the 24 h cycle in all of the studied months. There were significant variations in their peak timings and magnitudes over the year.

Effects of malathion on serum and glandular thyroxine (T 4 ), triiodothyronine (T 3 ), the T 3 T 4 ratio, peroxidase activity, and extrathyroidal conversion of T 4 to T 3 have been studied in the freshwater catfish Clarias batrachus during the prespawning and spawning phases of its annual reproductive cycle. Malathion inhibited T 4 secretion in the kidney, but accelerated T 4 synthesis in the pharyngeal thyroid. This pesticide also inhibited the extrathyroidal conversion of T 4 to T 3 in serum during both the studied phases.

Development of the flattened laminar structure in plant leaves requires highly regulated cell division and expansion patterns. Although tight regulation of these processes is essential during leaf development, leaf shape is highly diverse across the plant kingdom, implying that patterning of growth must be amenable to evolutionary change. Here, we describe the molecular identification of the classical tomato (Solanum lycopersicum) mutant lyrate, which is impaired in outgrowth of leaflet primodia and laminar tissues during compound leaf development. We found that the lyrate phenotype results from a loss-of-function mutation of the tomato JAGGED homolog, a well-described positive regulator of cell division in lateral organs. We demonstrate that LYRATE coordinates lateral outgrowth in the compound leaves of tomato by interacting with both the KNOX and auxin transcriptional networks and suggest that evolutionary changes in LYRATE expression may contribute to the fundamental difference between compound and simple leaves.

Freshwater female catfish, Clarias batrachus, were exposed to sublethal concentrations of carbaryl (12 mg/liter for 96 hr and 5 mg/liter for 16 days) during the prespawning and spawning phases of their annual reproductive cycle. The effects on the levels of thyroxine (T4) and triiodothyronine (T3), the T3/T4 ratio, and the extrathyroidal conversion of T4 to T3 were studied in serum, pharyngeal thyroid, and anterior and posterior kidney. Carbaryl exposure for 96 hr suppressed T4 but elevated T3 and the T3/T4 ratio in serum during both phases of experimentation. However, 16 days exposure caused a decline in T4 as well as in T3 and in the T3/T4 ratio. In the pharyngeal thyroid, carbaryl increased T4 as well as peroxidase activity and decreased T3 and the T3/T4 ratio. Its treatment for 96 hr did not elicit any change in the parameters studied in the anterior kidney, whereas in the posterior kidney it suppressed the levels of T4 and T3, peroxidase activity, and the T3/T4 ratio. However, carbaryl exposure for 16 days provoked increases in T4 and peroxidase activity but decreases in T3 and the T3/T4 ratio in anterior kidney. In posterior kidney, it did not change T4 and peroxidase activity, but decreased T3 and the T3/T4 ratio. It also stimulated extrathyroidal conversion of T4 to T3.

The application of genetic methods to the analysis of morphogenesis in maize is described. Several classes of floral mutants are differentiated through developmental studies and tests of epistasis. The results of mosaic and dosage analysis of Kn1, a dominant mutation affecting leaf development, are related to molecular studies of the gene.

Summary  It has been shown that the parasitic plant dodder (Cuscuta pentagona) establishes a continuous vascular system through which water and nutrients are drawn. Along with solutes, viruses and proteins, mRNA transcripts are transported from the host to the parasite. The path of the transcripts and their stability in the parasite have yet to be revealed.  To discover the route of mRNA transportation, the in situ reverse transcriptase– polymerase chain reaction (RT-PCR) technique was used to locally amplify host transcript within parasitic tissue. The stability of host mRNA molecules was also checked by monitoring specific transcripts along the growing dodder thread.  Four mRNAs, α and β subunits of PYROPHOSPHATE (PPi)-DEPENDENT PHOSPHOFRUCTOKINASE (LePFP), the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), and GIBBERELLIC ACID INSENSITIVE (LeGAI), were found to move from host (tomato (Solanum lycopersicum)) to dodder. LePFP mRNA was localized to the dodder parenchyma cells and to the phloem. LePFP transcripts were found in the growing dodder stem up to 30 cm from the tomato–dodder connection.  These results suggest that mRNA molecules are transferred from host to parasite via symplastic connections between parenchyma cells, move towards the phloem, and are stable for a long distance in the parasite. This may allow developmental coordination between the parasite and its host.

The present study, investigates the effect of RBx 6198, 2-{3-[4-(2-Isopropoxy-phenyl)-piperazin-1-yl]-propyl}-3a, 4, 7, 7a-tetrahydro-isoindole-1, 3,-dione, a novel alpha(1)-adrenoceptor antagonist, in both in vitro and in vivo test systems. RBx 6198 is a potent (nanomolar affinity) alpha(1A)-adrenoceptor antagonist with demonstrable uroselectivity in anaesthesized dog model. In radioligand binding studies using human recombinant receptors, RBx 6198 exhibited high selectivity (approximately 50 fold) for the alpha(1A)-adrenoceptor subtype as compared to alpha(1B)-adrenoceptor subtype. In order to assess tissue selectivity, the antagonistic effect of RBx 6198 on the phenylephrine induced contractile response of isolated rat prostate, spleen and aorta was characterized. RBx 6198 was 8 fold more potent in inhibiting phenylephrine-evoked contractions of isolated tissues compared to tamsulosin. However, the compound was non-selective for alpha(1A) vs. alpha(1D)-adrenoceptor like tamsulosin. In anaesthetized beagle dogs RBx 6198 suppressed the intraurethral pressure response to phenylephrine to a greater extent than the mean arterial pressure response thereby demonstrating uroselectivity consistent with in vitro binding and functional data. RBx 6198 was 6.4 fold more uroselective as compared to tamsulosin after i.v. route dose administration. Taken together all results from preclinical studies, it is suggested that RBx 6198 is a novel alpha(1)-adrenoceptor antagonist that exhibited improved pharmacological profile over tamsulosin in both in vitro and in vivo.

Across vascular plants, Class 1 KNOTTED1-like (KNOX1) genes appear to play a critical role in the development of compound leaves. An exception to this trend is found in the Fabaceae, where pea (Pisum sativum) uses UNIFOLIATA, an ortholog of the floral regulators FLORICAULA (FLO) and LEAFY (LFY), in place of KNOX1 genes to regulate compound leaf development. To assess the phylogenetic distribution of KNOX1-independent compound leaf development, a survey of KNOX1 protein expression across the Fabaceae was undertaken. The majority of compound-leafed Fabaceae have expression of KNOX1 proteins associated with developing compound leaves. However, in a large subclade of the Fabaceae, the inverted repeat–lacking clade (IRLC), of which pea is a member, KNOX1 expression is not associated with compound leaves. These data suggest that the FLO/LFY gene may function in place of KNOX1 genes in generating compound leaves throughout the IRLC. The contribution of FLO/LFY to leaf complexity in a member of the Fabaceae outside of the IRLC was examined by reducing expression of FLO/LFY orthologs in transgenic soybean (Glycine max). Transgenic plants with reduced FLO/LFY expression showed only slight reductions in leaflet number. Overexpression of a KNOX1 gene in alfalfa (Medicago sativa), a member of the IRLC, resulted in an increase in leaflet number. This implies that KNOX1 targets, which promote compound leaf development, are present in alfalfa and are still sensitive to KNOX1 regulation. These data suggest that KNOX1 genes and the FLO/LFY gene may have played partially overlapping roles in compound leaf development in ancestral Fabaceae but that the FLO/LFY gene took over this role in the IRLC.

The indeterminate shoot apical meristem of plants is characterized by the expression of the Class 1 KNOTTED1-LIKE HOMEOBOX ( KNOX1 ) genes. KNOX1 genes have been implicated in the acquisition and/or maintenance of meristematic fate. One of the earliest indicators of a switch in fate from indeterminate meristem to determinate leaf primordium is the down-regulation of KNOX1 genes orthologous to SHOOT MERISTEMLESS ( STM ) in Arabidopsis (hereafter called STM genes) in the initiating primordia. In simple leafed plants, this down-regulation persists during leaf formation. In compound leafed plants, however, KNOX1 gene expression is reestablished later in the developing primordia, creating an indeterminate environment for leaflet formation. Despite this knowledge, most aspects of how STM gene expression is regulated remain largely unknown. Here, we identify two evolutionarily conserved noncoding sequences within the 5′ upstream region of STM genes in both simple and compound leafed species across monocots and dicots. We show that one of these elements is involved in the regulation of the persistent repression and/or the reestablishment of STM expression in the developing leaves but is not involved in the initial down-regulation in the initiating primordia. We also show evidence that this regulation is developmentally significant for leaf formation in the pathway involving ASYMMETRIC LEAVES1/2 ( AS1/2 ) gene expression; these genes are known to function in leaf development. Together, these findings reveal a regulatory point of leaf development mediated through a conserved, noncoding sequence in STM genes.

Plant somatic cells have the remarkable ability to regenerate an entire organism. Many species in the genus Kalanchoë , known as “mother of thousands,” develop plantlets on the leaf margins. Using key regulators of organogenesis ( STM ) and embryogenesis ( LEC1 and FUS3 ) processes, we analyzed asexual reproduction in Kalanchoë leaves. Suppression of STM abolished the ability to make plantlets. Here, we report that constitutive plantlet-forming species, like Kalanchoë daigremontiana , form plantlets by coopting both organogenesis and embryogenesis programs into leaves. These species have a defective LEC1 gene and produce nonviable seed, whereas species that produce plantlets only upon stress induction have an intact LEC1 gene and produce viable seed. The latter species are basal in the genus, suggesting that induced-plantlet formation and seed viability are ancestral traits. We provide evidence that asexual reproduction likely initiated as a process of organogenesis and then recruited an embryogenesis program into the leaves in response to loss of sexual reproduction within this genus.

The Knotted-like homeobox (KNOX) genes play a profound role in shoot apical meristem organization, maintenance and leaf initiation. An MYB domain gene PHANTASTICA plays a role in regulating dorsal–ventral polarity in lateral organs and also downregulates a subset of the KNOX genes. Keywords: knotted-like; homeobox genes; KNOX; PHANTASTICA; plant development

www.sciencedirect.com The reviews on plant development presented here cover a wide range of subjects that seem only loosely connected. In a sense, this is a departure from the direction of the field over the past 15 years with its increasing focus on Arabidopsis as a model system. It is legitimate to ask whether we have lost something by narrowing our attention on a single species. Will Arabidopsis be like the Escherichia coli of the 1980s? Is it a powerful model system soon to be abandoned because it has made more complex systems accessible? Or to put it positively: does the remarkable progress made in Arabidopsis, combined with the ever increasing power of brute-force genomics, allow us to broaden our horizons again, revisit classical problems and perhaps identify novel avenues for research? While there is no sign at all of Arabidopsis losing its attractiveness, it is also true that alternative model species and comparative analyses are beginning to regain popularity. In this issue of Current Opinion in Plant Biology, we have attempted to provide examples of this new trend. In plant developmental biology, progress has been made on three fronts: comparative studies at the molecular and genetic level are illuminating the history of morphogenesis in plants; the evolution of key molecular players in developmental regulation is also being elucidated; and finally, developmental principles are being elucidated in the context of the whole organ or organism by utilizing real-time analyses and computer modeling.

A convenient and efficient synthesis of N6-substituted 3,6-diazabicyclo[3.2.1]octanes ( 6a–c ) has been achieved starting from suitably substituted lactams, which were converted to nitroenamines followed by reductive cyclization to afford 3,6-diazabicyclo[3.2.1]octane-2-ones in good yields. These bicyclic lactams were then reduced to the corresponding 3,6-diazabicyclo[3.2.1]octanes and converted to the required N3,N6-disubstituted 3,6-diazabicyclo[3.2.1]octanes ( 7a–h ), which were screened for α1-adrenoceptors antagonistic activities.

Plant development requires regulation of both cell division and differentiation. The class 1 KNOTTED1-like homeobox (KNOX) genes such as knotted1 (kn1) in maize (Zea mays) and SHOOTMERISTEMLESS in Arabidopsis (Arabidopsis thaliana) play a role in maintaining shoot apical meristem indeterminacy, and their misexpression is sufficient to induce cell division and meristem formation. KNOX overexpression experiments have shown that these genes interact with the cytokinin, auxin, and gibberellin pathways. The L1 layer has been shown to be necessary for the maintenance of indeterminacy in the underlying meristem layers. This work explores the possibility that the L1 affects meristem function by disrupting hormone transport pathways. The semidominant Extra cell layers1 (Xcl1) mutation in maize leads to the production of multiple epidermal layers by overproduction of a normal gene product. Meristem size is reduced in mutant plants and more cells are incorporated into the incipient leaf primordium. Thus, Xcl1 may provide a link between L1 division patterns, hormonal pathways, and meristem maintenance. We used double mutants between Xcl1 and dominant KNOX mutants and showed that Xcl1 suppresses the Kn1 phenotype but has a synergistic interaction with gnarley1 and rough sheath1, possibly correlated with changes in gibberellin and auxin signaling. In addition, double mutants between Xcl1 and crinkly4 had defects in shoot meristem maintenance. Thus, proper L1 development is essential for meristem function, and XCL1 may act to coordinate hormonal effects with KNOX gene function at the shoot apex.

A general method is reported for the synthesis of co-heterocyclic α-amino acids, which involves preparation of enaminone intermediates from thiolactams, followed by reaction with dinucleophiles, resulting in a single-stepcondensation and ring-chain transformation to the desired ω-heterocyclic α-amino acids after deprotection of amine and carboxylic group. The reaction steps preserve the chirality of the parent-substituted lactam. The method is illustrated by the synthesis of (2S)- and (2R)-2-amino-4-(1-aryl-/ 1,5-diaryl-1H-pyrazol-3-yl)butyric acids.