[Successful pembrolizumab therapy in metastasized adenosquamous carcinoma of the colon]. / Erfolgreiche Pembrolizumab-Therapie bei metastasiertem adenosquamösem Karzinom des Kolons.

Adenosquamous carcinoma (ASqC) is an exceedingly rare subtype of colorectal cancer without any known special guidelines for treatment. The biological behaviour and molecular background are widely unknown, although a few case studies report a worse prognosis compared to ordinary colorectal adenocarcinoma. We herein report for the first time the successful immune checkpoint inhibitor therapy in a 40-year-old patient suffering from metastasized right-sided colonic ASqC with unique molecular features, after having previously progressed under standard chemotherapy.

A detailed expression map of the PIN1 auxin transporter in Arabidopsis thaliana root.

BACKGROUND: Theauxin efflux carrier PIN1 is a key mediator of polar auxin transport in developing plant tissues. This is why factors that are supposed to be involved in auxin distribution are frequently tested in the regulation of PIN1 expression. As a result, diverse aspects of PIN1 expression are dispersed across dozens of papers entirely devoted to other specific topics related to the auxin pathway. Integration of these puzzle pieces about PIN1 expression revealed that, along with a recurring pattern, some features of PIN1 expression varied from article to article. To determine if this uncertainty is related to the specific foci of articles or has a basis in the variability of PIN1 gene activity, we performed a comprehensive 3D analysis of PIN1 expression patterns in Arabidopsis thaliana roots. RESULTS: We provide here a detailed map of PIN1 expression in the primary root, in the lateral root primordia and at the root-shoot junction. The variability in PIN1 expression pattern observed in individual roots may occur due to differences in auxin distribution between plants. To simulate this effect, we analysed PIN1 expression in the roots from wild type seedlings treated with different IAA concentrations and pin mutants. Most changes in PIN1 expression after exogenous IAA treatment and in pin mutants were also recorded in wild type but with lower frequency and intensity. Comparative studies of exogenous auxin effects on PIN1pro:GUS and PIN1pro:PIN1-GFP plants indicated that a positive auxin effect is explicit at the level of PIN1 promoter activity, whereas the inhibitory effect relates to post-transcriptional regulation. CONCLUSIONS: Our results suggest that the PIN1 expression pattern in the root meristem accurately reflects changes in auxin content. This explains the variability of PIN1 expression in the individual roots and makes PIN1 a good marker for studying root meristem activity.

Analysis of gene expression during parabolic flights reveals distinct early gravity responses in Arabidopsis roots.

Plant roots are among most intensively studied biological systems in gravity research. Altered gravity induces asymmetric cell growth leading to root bending. Differential distribution of the phytohormone auxin underlies root responses to gravity, being coordinated by auxin efflux transporters from the PIN family. The objective of this study was to compare early transcriptomic changes in roots of Arabidopsis thaliana wild type, and pin2 and pin3 mutants under parabolic flight conditions and to correlate these changes to auxin distribution. Parabolic flights allow comparison of transient 1-g, hypergravity and microgravity effects in living organisms in parallel. We found common and mutation-related genes differentially expressed in response to transient microgravity phases. Gene ontology analysis of common genes revealed lipid metabolism, response to stress factors and light categories as primarily involved in response to transient microgravity phases, suggesting that fundamental reorganisation of metabolic pathways functions upstream of a further signal mediating hormonal network. Gene expression changes in roots lacking the columella-located PIN3 were stronger than in those deprived of the epidermis and cortex cell-specific PIN2. Moreover, repetitive exposure to microgravity/hypergravity and gravity/hypergravity flight phases induced an up-regulation of auxin responsive genes in wild type and pin2 roots, but not in pin3 roots, suggesting a critical function of PIN3 in mediating auxin fluxes in response to transient microgravity phases. Our study provides important insights towards understanding signal transduction processes in transient microgravity conditions by combining for the first time the parabolic flight platform with the transcriptome analysis of different genetic mutants in the model plant, Arabidopsis.

Whole-mount in situ detection of microRNAs on Arabidopsis tissues using Zip Nucleic Acid probes.

MicroRNAs (miRNAs) affect fundamental processes of development. In plants miRNAs regulate organ development, transition to flowering, and responses to abiotic/biotic stresses. To understand the biological role of miRNAs, in addition to identifying their targeted transcripts, it is necessary to characterize the spatiotemporal regulation of their expression. Many methods have been used to define the set of organ-specific miRNAs by tissue dissection and miRNA profiling but none of them can describe their tissue and cellular distribution at the high resolution provided by in situ hybridization (ISH). This article describes the setup and optimization of a whole-mount ISH protocol to target endogenous miRNAs on intact Arabidopsis seedlings using DIG-labeled Zip Nucleic Acid (ZNA) oligonucleotide probes. Automation of the main steps of the procedure by robotized liquid handling has also been implemented in the protocol for best reproducibility of results, enabling running of ISH experiments at high throughput.

Modelling polar auxin transport in developmental patterning.

Auxin interacts with its own polar transport to influence cell polarity and tissue patterning. Research over the past decade has started to deliver new insights into the molecular mechanisms that drive and regulate polar auxin transport. The most prominent auxin efflux protein, PIN1, has subsequently become a crucial component of auxin transport models because it is now known to direct auxin flow and maintain local auxin gradients. Recent molecular and genetic experiments have allowed the formulation of conceptual models that are able to interpret the role of (i) auxin, (ii) its transport, and (iii) the dynamics of PIN1 in generating temporal and spatial patterns. Here we review the current mathematical models of patterning in two specific developmental contexts: lateral shoot and vein formation, focusing on how these models can help to untangle the details of auxin transport-mediated patterning.

The polycotyledon (pct1-2) mutant of tomato shows enhanced accumulation of PIN1 auxin transport facilitator protein.

The bilateral symmetry of a dicotyledon embryo is tightly associated with the directional flow of auxin. Disruption of polar auxin flow results in various developmental abnormalities. The pct1-2 mutant of tomato, showing polycotyledony, also has enhanced polar auxin transport in hypocotyls. Immunocytochemical analysis revealed increased PIN1 protein in pct1-2 roots and hypocotyls. The mutant also displayed an increase in PIN1 transcript levels in these organs. Our results indicate that over-accumulation of PIN1 protein is likely related to increased polar transport of auxin in the pct1-2 mutant.

Humic substances induce lateral root formation and expression of the early auxin-responsive IAA19 gene and DR5 synthetic element in Arabidopsis.

Humic substances (HS) have positive effects on plant physiology, but the molecular mechanisms underlying these events are only partially understood. HS exert auxin-like activity, but data supporting this hypothesis are under debate. To investigate the auxin-like activity of HS, we studied their biological effect on lateral root initiation in Arabidopsis thaliana. To this aim we characterised HS by means of DRIFT and (13)C CP/MAS NMR spectroscopy, and measured their endogenous content of IAA. We then utilised a combination of genetic and molecular approaches to unravel HS auxin activity in the initiation of lateral roots. The data obtained using specific inhibitors of auxin transport or action showed that HS induce lateral root formation mostly through their 'auxin activity'. These findings were further supported by the fact that HS used in this study activated the auxin synthetic reporter DR5::GUS and enhanced transcription of the early auxin responsive gene IAA19.

Auxin as a model for the integration of hormonal signal processing and transduction.

The regulation of plant growth responds to many stimuli. These responses allow environmental adaptation, thereby increasing fitness. In many cases, the relay of information about a plant's environment is through plant hormones. These messengers integrate environmental information into developmental pathways to determine plant shape. This review will use, as an example, auxin in the root of Arabidopsis thaliana to illustrate the complex nature of hormonal signal processing and transduction. It will then make the case that the application of a systems-biology approach is necessary, if the relationship between a plant's environment and its growth/developmental responses is to be properly understood.

Auxin transport and gravitational research: perspectives.

Gravity is a fundamental factor which affects all living organisms. Plant development is well adapted to gravity by directing roots downward and shoots upwards. For more than a century, plant biologists have been fascinated to describe the molecular mechanisms underlying the gravitropic response of plants. Important progress towards signal perception, transduction, and response has been made, but new tools are beginning to uncover the regulatory networks for gravitropic control. We summarise recent progress in study of gravitropism and discuss strategies to identify the molecular basis of the gravity response in Arabidopsis thaliana. This will put us on a road towards the molecular systems biology of the Arabidopsis gravitropic response.

Polar auxin transport in the wood-forming tissues of hybrid aspen is under simultaneous control of developmental and environmental signals.

Recent research has highlighted the importance of auxin concentration gradients during plant development. Establishment of these gradients is believed to involve polar auxin transport through specialized carrier proteins. We have used an experimental system, the wood-forming tissue of hybrid aspen, which allows tissue-specific expression analysis of auxin carrier genes and quantification of endogenous concentrations of the hormone. As part of this study, we isolated the putative polar auxin transport genes, PttLAX1-PttLAX3 and PttPIN1-PttPIN3, belonging to the AUX1-like family of influx and PIN1-like efflux carriers, respectively. Analysis of PttLAX and PttPIN expression suggests that specific positions in a concentration gradient of the hormone are associated with different stages of vascular cambium development and expression of specific members of the auxin transport gene families. We were also able demonstrate positive feedback of auxin on polar auxin transport genes. Entry into dormancy at the end of a growing season leads to a loss of auxin transport capacity, paralleled by reduced expression of PttLAX and PttPIN genes. Furthermore, data from field experiments show that production of the molecular components of the auxin transport machinery is governed by environmental controls. Our findings collectively demonstrate that trees have developed mechanisms to modulate auxin transport in the vascular meristem in response to developmental and environmental cues.

The putative sensor histidine kinase CKI1 is involved in female gametophyte development in Arabidopsis.

Embryo sac formation is a fundamental step in sexual reproduction in plants. However, the key players involved in the development of the female gametophyte remain elusive. We present data indicating that a two-component sensor histidine kinase, CKI1, originally implicated in cytokinin perception, is required for completion of megagametogenesis in Arabidopsis. We isolated a loss-of-function mutation in CKI1 resulting from an insertion of the En-1 transposon into the CKI1 coding sequence. Genetic analysis revealed that the mutant allele, cki1-i, could not be transmitted through the female germ line. Confocal laser scanning microscopy identified a block in megagametogenesis, characterized by the abortion of the central vacuole in mutant embryo sacs, and degradation of the developing female gametophyte after completion of all mitotic divisions. The recovery of two independent stable alleles and one revertant wild-type allele resulting from En-1 excision confirmed unambiguously the causal link between the cki1-i mutation and the abnormal phenotype. In situ localization of CKI1 mRNA and histochemical analysis of stable transformants harboring the uidA gene under the control of CKI1 promoter revealed that expression of CKI1 starts at the very beginning of female gametophyte development, and continues until fertilization. This suggests that the developing embryo sac may remain sensitive to signals recognized by CKI1 throughout megagametogenesis. Furthermore, expression of the paternally transmitted CKI1 was detected early after fertilization. The results indicate a role for a two-component signaling system during female gametophyte development, and provide the first evidence that gametophytic expression of a sensor-like molecule is essential for specific processes during megagametogenesis.

Mass spectrometric analysis reveals a cysteine bridge between residues 2 and 61 of the auxin-binding protein 1 from Zea mays L.

The major auxin-binding protein (ZmERabp1) from maize (Zea mays L.) has been structurally characterized. We determined the position of a disulfide bridge in ZmERabp1 by mass-spectrometric analysis. We show that Cys2 and Cys61 are covalently linked and that residue Cys155 bears the free sulfhydryl group. By making use of electrospray mass spectrometry, the molecular mass of ZmERabp1 was determined to be 20,243 Da comprising a sugar moiety of 1865 Da, corresponding to a high mannose-type glycan structure. Due to the high homology among all characterized ABPs, the information on the disulfide bonds will be important for functional analysis of recombinantly expressed ABP1.

Localization of the auxin permease AUX1 suggests two functionally distinct hormone transport pathways operate in the Arabidopsis root apex.

Auxins represent an important class of plant hormone that regulate plant development. Plants use specialized carrier proteins to transport the auxin indole-3-acetic acid (IAA) to target tissues. To date, efflux carrier-mediated polar auxin transport has been assumed to represent the sole mode of long distance IAA movement. Localization of the auxin permease AUX1 in the Arabidopsis root apex has revealed a novel phloem-based IAA transport pathway. AUX1, asymmetrically localized to the plasma membrane of root protophloem cells, is proposed to promote the acropetal, post-phloem movement of auxin to the root apex. MS analysis shows that IAA accumulation in aux1 mutant root apices is impaired, consistent with an AUX1 phloem unloading function. AUX1 localization to columella and lateral root cap tissues of the Arabidopsis root apex reveals that the auxin permease regulates a second IAA transport pathway. Expression studies using an auxin-regulated reporter suggest that AUX1 is necessary for root gravitropism by facilitating basipetal auxin transport to distal elongation zone tissues.

Auxin transport inhibitors block PIN1 cycling and vesicle trafficking.

Polar transport of the phytohormone auxin mediates various processes in plant growth and development, such as apical dominance, tropisms, vascular patterning and axis formation. This view is based largely on the effects of polar auxin transport inhibitors. These compounds disrupt auxin efflux from the cell but their mode of action is unknown. It is thought that polar auxin flux is caused by the asymmetric distribution of efflux carriers acting at the plasma membrane. The polar localization of efflux carrier candidate PIN1 supports this model. Here we show that the seemingly static localization of PIN1 results from rapid actin-dependent cycling between the plasma membrane and endosomal compartments. Auxin transport inhibitors block PIN1 cycling and inhibit trafficking of membrane proteins that are unrelated to auxin transport. Our data suggest that PIN1 cycling is of central importance for auxin transport and that auxin transport inhibitors affect efflux by generally interfering with membrane-trafficking processes. In support of our conclusion, the vesicle-trafficking inhibitor brefeldin A mimics physiological effects of auxin transport inhibitors.

The auxin signal for protoplast swelling is perceived by extracellular ABP1.

Protoplasts of corn coleoptiles and Arabidopsis hypocotyls respond to the plant hormone auxin with a rapid change in volume. We checked the effect of antibodies directed against epitopes of auxin-binding protein 1 from Arabidopsis thaliana (AtERabp1) and Zea mays (ZmERabp1), respectively. Antibodies raised against the C-terminus of AtERabp1 inhibited the response to auxin, while antibodies raised against a part of box a, the putative auxin-binding domain, induced a swelling response similar to that caused by auxin treatment. Synthetic C-terminal oligopeptides of ZmERabp1 also caused a swelling response. These effects occurred regardless of whether the experiments were carried out with homologous (anti-AtERabp1 antibodies on Arabidopsis protoplasts or anti-ZmERabp1 antibodies in maize protoplasts) or heterologous immunological tools. The results indicate that the auxin signal for protoplast swelling is perceived by extracellular ABP1.

BIG: a calossin-like protein required for polar auxin transport in Arabidopsis.

Polar auxin transport is crucial for the regulation of auxin action and required for some light-regulated responses during plant development. We have found that two mutants of Arabidopsis-doc1, which displays altered expression of light-regulated genes, and tir3, known for its reduced auxin transport-have similar defects and define mutations in a single gene that we have renamed BIG. BIG is very similar to the Drosophila gene Calossin/Pushover, a member of a gene family also present in Caenorhabditis elegans and human genomes. The protein encoded by BIG is extraordinary in size, 560 kD, and contains several putative Zn-finger domains. Expression-profiling experiments indicate that altered expression of multiple light-regulated genes in doc1 mutants can be suppressed by elevated levels of auxin caused by overexpression of an auxin biosynthetic gene, suggesting that normal auxin distribution is required to maintain low-level expression of these genes in the dark. Double mutants of tir3 with the auxin mutants pin1, pid, and axr1 display severe defects in auxin-dependent growth of the inflorescence. Chemical inhibitors of auxin transport change the intracellular localization of the auxin efflux carrier PIN1 in doc1/tir3 mutants, supporting the idea that BIG is required for normal auxin efflux.

Arabidopsis thaliana Rop GTPases are localized to tips of root hairs and control polar growth.

Plants contain a novel unique subfamily of Rho GTPases, vital components of cellular signalling networks. Here we report a general role for some members of this family in polarized plant growth processes. We show that Arabidopsis AtRop4 and AtRop6 encode functional GTPases with similar intrinsic GTP hydrolysis rates. We localized AtRop proteins in root meristem cells to the cross-wall and cell plate membranes. Polar localization of AtRops in trichoblasts specifies the growth sites for emerging root hairs. These sites were visible before budding and elongation of the Arabidopsis root hair when AtRops accumulated at their tips. Expression of constitutively active AtRop4 and AtRop6 mutant proteins in root hairs of transgenic Arabidopsis plants abolished polarized growth and delocalized the tip-focused Ca2+ gradient. Polar localization of AtRops was inhibited by brefeldin A, but not by other drugs such as latrunculin B, cytochalasin D or caffeine. Our results demonstrate a general function of AtRop GTPases in tip growth and in polar diffuse growth.

Bus, a bushy Arabidopsis CYP79F1 knockout mutant with abolished synthesis of short-chain aliphatic glucosinolates.

A new mutant of Arabidopsis designated bus1-1 (for bushy), which exhibited a bushy phenotype with crinkled leaves and retarded vascularization, was characterized. The phenotype was caused by an En-1 insertion in the gene CYP79F1. The deduced protein belongs to the cytochrome P450 superfamily. Because members of the CYP79 subfamily are believed to catalyze the oxidation of amino acids to aldoximes, the initial step in glucosinolate biosynthesis, we analyzed the level of glucosinolates in a CYP79F1 null mutant (bus1-1f) and in an overexpressing plant. Short-chain glucosinolates derived from methionine were completely lacking in the null mutant and showed increased levels in the overexpressing plant, indicating that CYP79F1 uses short-chain methionine derivatives as substrates. In addition, the concentrations of indole-3-ylmethyl-glucosinolate and the content of the auxin indole-3-acetic acid and its precursor indole-3-acetonitrile were increased in the bus1-1f mutant. Our results demonstrate for the first time that the formation of glucosinolates derived from methionine is mediated by CYP79F1 and that knocking out this cytochrome P450 has profound effects on plant growth and development.

KAT1 is not essential for stomatal opening.

It is generally accepted that K(+) uptake into guard cells via inward-rectifying K(+) channels is required for stomatal opening. To test whether the guard cell K(+) channel KAT1 is essential for stomatal opening, a knockout mutant, KAT1En-1, was isolated from an En-1 mutagenized Arabidopsis thaliana population. Stomatal action and K(+) uptake, however, were not impaired in KAT1-deficient plants. Reverse transcription-PCR experiments with isolated guard cell protoplasts showed that in addition to KAT1, the K(+) channels AKT1, AKT2/3, AtKC1, and KAT2 were expressed in this cell type. In impalement measurements, intact guard cells exhibited inward-rectifying K(+) currents across the plasma membrane of both wild-type and KAT1En-1 plants. This study demonstrates that multiple K(+) channel transcripts exist in guard cells and that KAT1 is not essential for stomatal action.