GENETIC DISSECTION OF DEVELOPMENTAL TRAITS IN BARLEY (HORDEUM VULGARE)

Barley (Hordeum vulgare) ranks in fourth place among cultivated cereals for worldwide production and is a recognized model organism for genetic and genomic studies in the Triticeae tribe, which includes wheats (Triticum species) and rye (Secale cereale). Root and shoot architecture traits are key factors in plant performance, competition with weeds, adaptation and stress responses thus having an important impact on yield and yield stability. Breeders have proposed hypothetical optimal morphological parameters to improve production in relation to different environmental conditions. Leaf size and orientation are determinants of canopy transpiration and radiation interception e.g. in dry and sunny Mediterranean environments reduced size and erect orientation of the leaves can reduce water loss by transpiration and allow deeper light penetration into the canopy. Tillering influences crop performance, biomass and grain production, e.g. a reduction in tillering compensated by an increase dimension and number of kernels per spike could be a strategy of adaptation to dry climates. A reduction in plant height and an augment in stem thickness is connected to lodging resistance. Root system extension is connected to the ability of the plant to reach water. The objectives of this project were to dissect genetic variability for shoot and root morphological traits in barley, identifying genomic regions and characterizing genes controlling these traits, and exploring how different traits influence each other. To this end, two approaches were undertaken depending on the trait(s) under study: \u2022 the first exploited natural variation in a panel of modern and old European barley cultivars to carry out association mapping of flowering date, stem diameter, spike fertility, leaf dimension, plant height, tillering and root extension (Chapters 2 and 3); \u2022 the second was to characterize the ontogenetic basis of increased tillering using as a case study the many-noded dwarf6.6 (mnd6.6) high tillering barley mutant (Chapter 4). In the first approach, we focused on winter barley because of its agronomic interest in the Mediterranean area, where genetic improvement of drought tolerance is particularly important. We analyzed a panel of 142 European winter barley cultivars (67 two-rowed and 75 six-rowed) with a view to conduct a genome wide association scan (GWAS) for shoot and root architecture traits in two separate sets of experiments. To this end, genotyping data for 4,083 SNPs were available from previous projects of which 2,521 mapped on the POPSEQ barley reference map. PCoA results indicated the existence of two major sub-populations in our germplasm panel, corresponding to two-rowed and six-rowed barley cultivars. In order to study shoot developmental traits (Chapter 2) the panel was phenotyped during the growing season 2012-2013 in a field trial at Fiorenzuola d\u2019Arda, Piacenza, Italy. The experimental scheme consisted in 3 replicates (each being a plot of 24 well spaced plants) in randomized blocks. For selected traits data were integrated and analyzed together with those coming from a parallel field trial that was carried out at the University of Shiraz, Iran (data courtesy of Dr. Elahe Tavakol). Flowering date (FD) and leaf width (LW) were measured in both Italy and Iran, leaf length (LL) was measured only in Iran, plant height (PH), spike length (SL), number of fertile rachis node per spike (NFRN) tillering (T) and (SD) were measured only in Italy. Best Linear Unbiased Estimators (BLUEs) of FD, LW were calculated as the phenotypic values estimated for each genotype in a mixed linear model, where genotypes were set as fixed factor and location, location-genotype interaction and replicates as random factors. For BLUEs calculation of all other traits only replicates were used as random factors. BLUEs were subjected to GWAS analyses, using a mixed linear model (MLM) correcting for population structure with a Q matrix (PCA first three coordinate) and for individuals co-ancestry using a K matrix (a pair-wise matrix defining the degree of genetic covariance among individuals). Significance of marker-trait associations was evaluated based on false discovery rate (FDR)-adjusted p-values (threshold value for significant association was set at 0.05). All traits except tillering exhibited good heritability. Few QTLs were detected in GWAS (five for FD, two for LW, three for LL, one for PH, two for SL, two for NFRN, one for tillering, no one for SM). Flowering date exhibited significant correlation with leaf dimension and spike length and six markers designed on Photoperiod-H1 (Ppd-H1) gene (the major determinant for photoperiod response in barley) were the most significantly associated to FD, LW, LL and SL. In particular the recessive ppd- H1 allele causing reduced photoperiod sensitivity, delayed flowering date and increased leaf dimension and spike length compared to the Ppd-H1 allele. Three markers diagnostic for the HvCEN gene (which regulates flowering date independently from photoperiod) were significantly associated to FD and SL. These results suggested that genes for flowering date could have pleiotropic effects on other morphological traits that may mask other genetic effects. For this reason we tested a novel approach repeating GWAS for LW, LL and SL using flowering date as a cofactor (fixed effect) in further analyses. For SL and LW no new significant associations were found with this method, while new significant associations were uncovered for LL, including two markers on chromosome 5H mapped in a region where narrow leaf dwarf 1a (nld-1a) mutant had previously been previously mapped. Tillering and NFRN were only associated to markers diagnostic INTERMEDIUM-C (INT-C), one of the two main genes controlling row type: in our panel two-rowed genotypes had a significantly higher number of tillers and NFRN compared to six- rowed varieties, confirming the known pleiotropic effects of row-type genes on tillering and NFRN and the balancing of patterns of development by breeding practice for the particular row-type. Based on these results, we run GWAS for NFRN and tillering using row-type as covariate. With this model, we found six markers associated with NFRN on chromosome 5H, in the region hosting HvCO12, HvCO13, HvCO15, XvCCA-1, HvLHY, genes involved in control of flowering date. These same markers, were associated to the duration of the phase between awn primordia formation and tipping (awn arising from flag leaf) in a recently published GWAS study. Together, results from Chapter 2 provide the first evidence of the involvement of the Ppd-H1 gene in control of leaf size and spike length. Thus few QTLs were detected that explain the phenotypic variation for our morphological traits, with some major genes having strong pleiotropic effects that mask minor genetic effects. The use of traits that appear to influence others measures as covariates in GWAS models seems to be a promising approach, although the statistical power of this strategy is still to be evaluated. Germplasm collections with uniform growth habit and row-type are an attractive alternative to prevent confounding effects and allow additional loci to be detected. In Chapter 3, we explored natural genetic variation in root extension using the same winter barley panel as Chapter 2 in growth chamber experiments. In order to evaluate root growth we built 50 cm deep cylindrical pots (called rhizotrons) and used digital scans of the root system to measure total root extension with the winRHIZO software. Based on a series of preliminary tests, we used siliceous sand supplemented with controlled release fertilizer to analyze 4th leaf stage plants from 31 genotypes (9 plants per genotype). Root extension per se exhibited 75% heritability, while normalizing root extension on shoot dry weight resulted in low variability (22%) likely due to low heritability of shoot dry weight in our system. These results support the validity of our protocol for evaluation of genetic variation in root extension in barley and other cereals and indicate significant variation exists in our germplasm panel. Thus, the already collected material will be analysed to phenotype the entire panel. In the future, more variability may be uncovered by exploring wild barleys (Hordeum vulgare spp. spontaneum) or landraces. Tillering is a plastic trait affected by the complex interplay of genetic and hormonal factors with environmental conditions such as plant density/light quality and nutrient availability, which likely complicated genetic dissection of this trait in our field experiment on the winter barley panel (Chapter 2). To circumvent the limited power of the GWAS approach for this trait and understand more about the mechanisms subtending tiller formation, we decided to use the mnd6.6 mutant as a case study to investigate the ontogenetic basis of high tillering in barley and its relation to leaf development. Mutant and wild-type plants were grown in growth in a controlled chamber under long day conditions, and dissected weekly from the emergence to anthesis, registering the development of axillary buds, leaves and tillers together with internode elongation, in relation to shoot apical meristem (SAM) stage. Results show that the mutant is not altered in timing of apical meristem development and differentiation to spike, but has a shorter phyllochron that leads to an increment in the number of leaves per vegetative axis. This in turn results in a higher number of axillary buds and a higher number of tillers. The HvMND6 gene was recently identified and our results are consistent with the activity of the previously characterized rice homologue PLASTOCHRON1, indicating an evolutionarily conserved link between plastochron/phyllochron duration and tillering. Concluding, while significant genetic variation was identified for various traits within the gene pool of our winter barley collection, variability of morphological traits as leaf dimension was subordinated to the length of vegetative period. Indeed, flowering date is one of the major factors on which breeding practice has worked to adapt barley to different environments. Beyond modern European varieties, barley breeding for new ideotypes should explore wider genetic resources as Hordeum spp. spontaneum or landraces. In any case, the existence of correlations between different phenotypes calls for careful evaluation of sources of traits to avoid undesired effects on other traits, e.g. due to the relation between tillering and phyllochrone, breeding for early plant vigour through shortening phyllochron, may have pleiotropic effects and result in increased tillering whose benefits would have to be evaluated

Proteomic profiling reveals the transglutaminase-2 externalization pathway in kidneys after unilateral ureteric obstruction

Increased export of transglutaminase-2 (TG2) by tubular epithelial cells (TECs) into the surrounding interstitium modifies the extracellular homeostatic balance, leading to fibrotic membrane expansion. Although silencing of extracellular TG2 ameliorates progressive kidney scarring in animal models of CKD, the pathway through which TG2 is secreted from TECs and contributes to disease progression has not been elucidated. In this study, we developed a global proteomic approach to identify binding partners of TG2 responsible for TG2 externalization in kidneys subjected to unilateral ureteric obstruction (UUO) using TG2 knockout kidneys as negative controls. We report a robust and unbiased analysis of the membrane interactome of TG2 in fibrotic kidneys relative to the entire proteome after UUO, detected by SWATH mass spectrometry. The data have been deposited to the ProteomeXchange with identifier PXD008173. Clusters of exosomal proteins in the TG2 interactome supported the hypothesis that TG2 is secreted by extracellular membrane vesicles during fibrosis progression. In established TEC lines, we found TG2 in vesicles of both endosomal (exosomes) and plasma membrane origin (microvesicles/ectosomes), and TGF-β1 stimulated TG2 secretion. Knockout of syndecan-4 (SDC4) greatly impaired TG2 exosomal secretion. TG2 coprecipitated with SDC4 from exosome lysate but not ectosome lysate. Ex vivo, EGFP-tagged TG2 accumulated in globular elements (blebs) protruding/retracting from the plasma membrane of primary cortical TECs, and SDC4 knockout impaired bleb formation, affecting TG2 release. Through this combined in vivo and in vitro approach, we have dissected the pathway through which TG2 is secreted from TECs in CKD

Spotlight on the transglutaminase 2-heparan sulfate interaction

Heparan sulfate proteoglycans (HSPGs), syndecan-4 (Sdc4) especially, have been suggested as potential partners of transglutaminase-2 (TG2) in kidney and cardiac fibrosis, metastatic cancer, neurodegeneration and coeliac disease. The proposed role for HSPGs in the trafficking of TG2 at the cell surface and in the extracellular matrix (ECM) has been linked to the fibrogenic action of TG2 in experimental models of kidney fibrosis. As the TG2-HSPG interaction is largely mediated by the heparan sulfate (HS) chains of proteoglycans, in the past few years a number of studies have investigated the affinity of TG2 for HS, and the TG2 heparin binding site has been mapped with alternative outlooks. In this review, we aim to provide a compendium of the main literature available on the interaction of TG2 with HS, with reference to the pathological processes in which extracellular TG2 plays a role

An extracellular transglutaminase is required for apple pollen tube growth

An extracellular form of the calcium-dependent protein-crosslinking enzyme TGase (transglutaminase) was demonstrated to be involved in the apical growth of Malus domestica pollen tube. Apple pollen TGase and its substrates were co-localized within aggregates on the pollen tube surface, as determined by indirect immunofluorescence staining and the in situ cross-linking of fluorescently labelled substrates. TGase-specific inhibitors and an anti-TGase monoclonal antibody blocked pollen tube growth, whereas incorporation of a recombinant fluorescent mammalian TGase substrate (histidine-tagged green fluorescent protein:His6– Xpr–GFP) into the growing tube wall enhanced tube length and germination, consistent with a role of TGase as a modulator of cell wall building and strengthening. The secreted pollen TGase catalysed the cross-linking of both PAs (polyamines) into proteins (released by the pollen tube) and His6-Xpr-GFP into endogenous or exogenously added substrates. A similar distribution of TGase activitywas observed in planta on pollen tubes germinating inside the style, consistent with a possible additional role for TGase in the interaction between the pollen tube and the style during fertilization

Genome-Wide Association Mapping of Root Extension in a Collection of European Winter Barley Cultivars

Root extension in cereals is an extremely plastic trait exhibiting high variation in relation to the genetic background and to environmental conditions. The study of root system is particularly important in the Mediterranean area, where genetic improvement of drought tolerance on winter barley is a relevant breeding target. Here we aimed at exploring the natural genetic variation in root extension in a collection of European winter barley cultivars (67 two-rowed and 75 six-rowed, released between 1921 and 2006). For each genotype, three plants were grown in cylindrical pots (rhizotrons) with diameter of 10 cm and 50 cm height, filled with siliceous sand. Plants were collected at the 4 leaf stage (Zadocks stage 14), when roots were separated from shoots and scanned. The obtained images were analyzed by using the winRHIZO software to calculate the total root extension, as the sum of lengths of primary and secondary roots. The whole experiment was replicated three times, showing repeatability of 0.53. The same collection was previously genotyped for >7000 iSelect SNP markers, providing a powerful tool for association mapping of root traits. Genotype-phenotype association with the R-GAPIT package identified a significant genomic region on chromosome 5H-bin7, that has been scrutinized for candidate genes and alleles with a putative role in the trait under study

Involvement of the HLXB9 homeobox gene in Currarino syndrome [2]

published_or_final_versio

Astrocytes-derived extracellular vesicles in motion at the neuron surface: Involvement of the prion protein

Astrocytes-derived extracellular vesicles (EVs) are key players in glia-neuron communication. However, whether EVs interact with neurons at preferential sites and how EVs reach these sites on neurons remains elusive. Using optical manipulation to study single EV-neuron dynamics, we here show that large EVs scan the neuron surface and use neuronal processes as highways to move extracellularly. Large EV motion on neurites is driven by the binding of EV to a surface receptor that slides on neuronal membrane, thanks to actin cytoskeleton rearrangements. The use of prion protein (PrP)-coated synthetic beads and PrP knock out EVs/neurons points at vesicular PrP and its receptor(s) on neurons in the control of EV motion. Surprisingly, a fraction of large EVs contains actin filaments and has an independent capacity to move in an actin-mediated way, through intermittent contacts with the plasma membrane. Our results unveil, for the first time, a dual mechanism exploited by astrocytic large EVs to passively/actively reach target sites on neurons moving on the neuron surface

Tissue carcinoembryonic antigen and oestrogen receptor status in breast carcinoma: an immunohistochemical study of clinical outcome in a series of 252 patients with long-term follow-up.

Carcinoembryonic antigen (CEA) is a well-known tumour marker whose immunohistochemical expression could be prognostically relevant in breast carcinomas. We evaluated CEA immunohistochemical expression, using the specific T84.66 monoclonal antibody, in a series of 252 consecutive cases of infiltrating breast carcinomas (104 N0, 148 N1/2) with median follow-up of 84 months. Oestrogen receptor (ER) status has been evaluated with the immunohistochemical method (ER1D5 antibody, 10% cut-off value): 121 cases were ER negative, 128 cases were ER positive and in three cases ER status was unknown. CEA staining was cytoplasmic; staining intensity and percentage of reacting cells were combined to obtain a final score (CEA score). The difference between the distribution of CEA score within the modalities of the other variables was not statistically significant. Univariate survival analysis has been performed on the series of node-negative and node-positive patients. In the latter subgroup, this has been performed separately for patients treated with systemic adjuvant hormonal therapy or chemotherapy. A multivariate analysis was only performed for node-positive patients treated with adjuvant therapy. CEA immunoreactivity was not prognostically relevant in any subset of analysed patients. The most important prognostic markers were nodal status and tumour size