CD163+ macrophages in mantle cell lymphoma induce activation of pro-survival pathways and immune suppression.

Mantle cell lymphoma (MCL) is dependent on a supportive tumor immune microenvironment (TIME), where infiltration of CD163+ macrophages has a negative prognostic impact. This study explores how abundance and spatial localization of CD163+ cells are associated with the biology of the MCL TIME. This is achieved through spatial multi-omic investigations of tumor and infiltrating CD163+ and CD3+ cells, respectively. We analyzed diagnostic MCL tissue from 100 patients. Sixty-three proteins were measured by GeoMx® digital spatial profiling in tissue microarrays. Regions of interests (ROIs) were selected in tumor-rich and tumor-sparse tissue regions. Molecular profiling of CD163+ macrophage segments, CD20+ MCL tumor cell segments and CD3+ T-cell segments was performed. To validate protein profiles, 1811 mRNAs were measured in CD20+ cells and two subsets of T-cells. Image analysis was used to extract the phenotype and position of each targeted cell allowing exploration of cell frequencies and cellular neighborhoods. Proteomic investigations revealed that CD163+ cells modulate their immune profile depending on the localization and that the immune inhibitory molecules VISTA and B7-H3 have higher expression in tumor-sparse versus tumor-rich tissue regions and targeting should be explored. We show that MCL tissues with more abundant infiltration of CD163+ cells have a higher expression of key components of the mitogen-activated protein kinase (MAPK) pathway, which was validated by complementary mRNA analyses. Thus, the MAPK pathway may be a feasible therapeutic target in MCL patients with CD163+ cell infiltration. We further show the independent and combined prognostic value of CD11c and CD163 beyond established risk factors.

The role of the testa during the establishment of physical dormancy in the pea seed.

BACKGROUND: A water-impermeable testa acts as a barrier to a seed's imbibition, thereby imposing dormancy. The physical and functional properties of the macrosclereids are thought to be critical determinants of dormancy; however, the mechanisms underlying the maintenance of and release from dormancy in pea are not well understood. METHODS: Seeds of six pea accessions of contrasting dormancy type were tested for their ability to imbibe and the permeability of their testa was evaluated. Release from dormancy was monitored following temperature oscillation, lipid removal and drying. Histochemical and microscopic approaches were used to characterize the structure of the testa. KEY RESULTS: The strophiole was identified as representing the major site for the entry of water into non-dormant seeds, while water entry into dormant seeds was distributed rather than localized. The major barrier for water uptake in dormant seeds was the upper section of the macrosclereids, referred to as the 'light line'. Dormancy could be released by thermocycling, dehydration or chloroform treatment. Assays based on either periodic acid or ruthenium red were used to visualize penetration through the testa. Lipids were detected within a subcuticular waxy layer in both dormant and non-dormant seeds. The waxy layer and the light line both formed at the same time as the establishment of secondary cell walls at the tip of the macrosclereids. CONCLUSIONS: The light line was identified as the major barrier to water penetration in dormant seeds. Its outer border abuts a waxy subcuticular layer, which is consistent with the suggestion that the light line represents the interface between two distinct environments - the waxy subcuticular layer and the cellulose-rich secondary cell wall. The mechanistic basis of dormancy break includes changes in the testa's lipid layer, along with the mechanical disruption induced by oscillation in temperature and by a decreased moisture content of the embryo.

The dehydration stress of couch grass is associated with its lipid metabolism, the induction of transporters and the re-programming of development coordinated by ABA.

BACKGROUND: The wild relatives of crop species represent a potentially valuable source of novel genetic variation, particularly in the context of improving the crop's level of tolerance to abiotic stress. The mechanistic basis of these tolerances remains largely unexplored. Here, the focus was to characterize the transcriptomic response of the nodes (meristematic tissue) of couch grass (a relative of barley) to dehydration stress, and to compare it to that of the barley crown formed by both a drought tolerant and a drought sensitive barley cultivar. RESULTS: Many of the genes up-regulated in the nodes by the stress were homologs of genes known to be mediated by abscisic acid during the response to drought, or were linked to either development or lipid metabolism. Transporters also featured prominently, as did genes acting on root architecture. The resilience of the couch grass node arise from both their capacity to develop an altered, more effective root architecture, but also from their formation of a lipid barrier on their outer surface and their ability to modify both their lipid metabolism and transporter activity when challenged by dehydration stress. CONCLUSIONS: Our analysis revealed the nature of dehydration stress response in couch grass. We suggested the tolerance is associated with lipid metabolism, the induction of transporters and the re-programming of development coordinated by ABA. We also proved the applicability of barley microarray for couch grass stress-response analysis.

Towards Better Understanding of Pea Seed Dormancy Using Laser Desorption/Ionization Mass Spectrometry.

Seed coats of six pea genotypes contrasting in dormancy were studied by laser desorption/ionization mass spectrometry (LDI-MS). Multivariate statistical analysis discriminated dormant and non-dormant seeds in mature dry state. Separation between dormant and non-dormant types was observed despite important markers of particular dormant genotypes differ from each other. Normalized signals of long-chain hydroxylated fatty acids (HLFA) in dormant JI64 genotype seed coats were significantly higher than in other genotypes. These compounds seem to be important markers likely influencing JI64 seed imbibition and germination. HLFA importance was supported by study of recombinant inbred lines (JI64xJI92) contrasting in dormancy but similar in other seed properties. Furthemore HLFA distribution in seed coat was studied by mass spectrometry imaging. HLFA contents in strophiole and hilum are significantly lower compared to other parts indicating their role in water uptake. Results from LDI-MS experiments are useful in understanding (physical) dormancy (first phases of germination) mechanism and properties related to food processing technologies (e.g., seed treatment by cooking).

A Combined Comparative Transcriptomic, Metabolomic, and Anatomical Analyses of Two Key Domestication Traits: Pod Dehiscence and Seed Dormancy in Pea (Pisum sp.).

The origin of the agriculture was one of the turning points in human history, and a central part of this was the evolution of new plant forms, domesticated crops. Seed dispersal and germination are two key traits which have been selected to facilitate cultivation and harvesting of crops. The objective of this study was to analyze anatomical structure of seed coat and pod, identify metabolic compounds associated with water-impermeable seed coat and differentially expressed genes involved in pea seed dormancy and pod dehiscence. Comparative anatomical, metabolomics, and transcriptomic analyses were carried out on wild dormant, dehiscent Pisum elatius (JI64, VIR320) and cultivated, indehiscent Pisum sativum non-dormant (JI92, Cameor) and recombinant inbred lines (RILs). Considerable differences were found in texture of testa surface, length of macrosclereids, and seed coat thickness. Histochemical and biochemical analyses indicated genotype related variation in composition and heterogeneity of seed coat cell walls within macrosclereids. Liquid chromatography-electrospray ionization/mass spectrometry and Laser desorption/ionization-mass spectrometry of separated seed coats revealed significantly higher contents of proanthocyanidins (dimer and trimer of gallocatechin), quercetin, and myricetin rhamnosides and hydroxylated fatty acids in dormant compared to non-dormant genotypes. Bulk Segregant Analysis coupled to high throughput RNA sequencing resulted in identification of 770 and 148 differentially expressed genes between dormant and non-dormant seeds or dehiscent and indehiscent pods, respectively. The expression of 14 selected dormancy-related genes was studied by qRT-PCR. Of these, expression pattern of four genes: porin (MACE-S082), peroxisomal membrane PEX14-like protein (MACE-S108), 4-coumarate CoA ligase (MACE-S131), and UDP-glucosyl transferase (MACE-S139) was in agreement in all four genotypes with Massive analysis of cDNA Ends (MACE) data. In case of pod dehiscence, the analysis of two candidate genes (SHATTERING and SHATTERPROOF) and three out of 20 MACE identified genes (MACE-P004, MACE-P013, MACE-P015) showed down-expression in dorsal and ventral pod suture of indehiscent genotypes. Moreover, MACE-P015, the homolog of peptidoglycan-binding domain or proline-rich extensin-like protein mapped correctly to predicted Dpo1 locus on PsLGIII. This integrated analysis of the seed coat in wild and cultivated pea provides new insight as well as raises new questions associated with domestication and seed dormancy and pod dehiscence.

Global Scale Transcriptional Profiling of Two Contrasting Barley Genotypes Exposed to Moderate Drought Conditions: Contribution of Leaves and Crowns to Water Shortage Coping Strategies.

Drought is a serious threat for sustainable agriculture. Barley represents a species well adapted to environmental stresses including drought. To elucidate the adaptive mechanism of barley on transcriptional level we evaluated transcriptomic changes of two contrasting barley cultivars upon drought using the microarray technique on the level of leaves and crowns. Using bioinformatic tools, differentially expressed genes in treated vs. non-treated plants were identified. Both genotypes revealed tissue dehydration under drought conditions as shown at water saturation deficit and osmotic potential data; however, dehydration was more severe in Amulet than in drought-resistant Tadmor under the same ambient conditions. Performed analysis showed that Amulet enhanced expression of genes related to active plant growth and development, while Tadmor regarding the stimulated genes revealed conservative, water saving strategy. Common reactions of both genotypes and tissues included an induction of genes encoding several stress-responsive signaling proteins, transcription factors as well as effector genes encoding proteins directly involved in stress acclimation. In leaf, tolerant cultivar effectively stimulated mainly the expression of genes encoding proteins and enzymes involved in protein folding, sulfur metabolism, ROS detoxification or lipid biosynthesis and transport. The crown specific reaction of tolerant cultivar was an enhanced expression of genes encoding proteins and enzymes involved in cell wall lignification, ABRE-dependent abscisic acid (ABA) signaling, nucleosome remodeling, along with genes for numerous jasmonate induced proteins.

The up-regulation of elongation factors in the barley leaf and the down-regulation of nucleosome assembly genes in the crown are both associated with the expression of frost tolerance.

We report a series of microarray-based leaf and crown transcriptome comparisons involving three barley cultivars (cvs. Luxor, Igri and Atlas 68) which express differing degrees of frost tolerance. The transcripts were obtained following the exposure of seedlings to low (above and below zero) temperatures, aiming to identify those genes and signalling/metabolic pathways which are associated with frost tolerance. Both the leaves and the crowns responded to low temperature by the up-regulation of a suite of abscisic acid (ABA)-responsive genes, most of which have already been recognized as components of the plant low temperature response. The inter-cultivar comparison indicated that genes involved in maintaining the leaf's capacity to synthesize protein and to retain chloroplast activity were important for the expression of frost tolerance. In the crown, the repression of genes associated with nucleosome assembly and transposon regulation were the most relevant transcriptional changes associated with frost tolerance, highlighting the role of gene repression in the cold acclimation response.

The choice of reference gene set for assessing gene expression in barley (Hordeum vulgare L.) under low temperature and drought stress.

Drought and low temperature are the two most significant causes of abiotic stress in agricultural crops and, therefore, they pose considerable challenges in plant science. Hence, it is crucial to study response mechanisms and to select genes for identification signaling pathways that lead from stimulus to response. The assessment of gene expression is often attempted using real-time RT-PCR (qRT-PCR), a technique which requires a careful choice of reference gene(s) for normalization purpose. Here, we report a comparison of 13 potential reference genes for studying gene expression in the leaf and crown of barley seedlings subjected to low temperature or drought stress. All three currently available software packages designed to identify reference genes from qRT-PCR data (GeNorm, NormFinder and BestKeeper) were used to identify informative sets of up to three reference genes. Interestingly, the data obtained from the separate treatment of leaf and crown have led to the recommendations that HSP70 and S-AMD (and possibly HSP90) to be used as the reference genes for low-temperature stressed leaves, HSP90 and EF1α for low-temperature stressed crowns, cyclophilin and ADP-RF (and possibly ACT) for drought-stressed leaves, and EF1α and S-AMD for drought-stressed crowns. Our results have demonstrated that the gene expression can be highly tissue- or organ-specific in barley and have confirmed that reference gene choice is essential in qRT-PCR. The findings can also serve as guidelines for the selection of reference genes under different stress conditions and lay foundation for more accurate and widespread use of qRT-PCR in barley gene analysis.

Transcriptional responses of winter barley to cold indicate nucleosome remodelling as a specific feature of crown tissues.

We report a series of microarray-based comparisons of gene expression in the leaf and crown of the winter barley cultivar Luxor, following the exposure of young plants to various periods of low (above and below zero) temperatures. A transcriptomic analysis identified genes which were either expressed in both the leaf and crown, or specifically in one or the other. Among the former were genes responsible for calcium and abscisic acid signalling, polyamine synthesis, late embryogenesis abundant proteins and dehydrins. In the crown, the key organ for cereal overwintering, cold treatment induced transient changes in the transcription of nucleosome assembly genes, and especially H2A and HTA11, which have been implicated in cold sensing in Arabidopsis thaliana. In the leaf, various heat-shock proteins were induced. Differences in expression pattern between the crown and leaf were frequent for genes involved in certain pathways responsible for osmolyte production (sucrose and starch, raffinose, γ-aminobutyric acid metabolism), sugar signalling (trehalose metabolism) and secondary metabolism (lignin synthesis). The action of proteins with antifreeze activity, which were markedly induced during hardening, was demonstrated by a depression in the ice nucleation temperature.