A gene encoding a SHINE1/WAX INDUCER1 transcription factor controls cuticular wax in barley

All land plants seal their above ground body parts with a lipid-rich hydrophobic barrier called the cuticle to protect themselves from dehydration and other terrestrial threats. Mutational studies in several model species have identified multiple loci regulating cuticular metabolism and development. Of particular importance are the eceriferum (cer) mutants characterized by a loss of cuticular wax. Some barley cer mutants, including cer-x, show defects in the distinctive β-diketone-enriched wax bloom on reproductive stage leaf sheaths, stems, and spikes. We exploited extensive allelic populations, near-isogenic lines, and powerful genotyping platforms to identify variation in the HvWAX INDUCER1 (HvWIN1) gene, encoding a SHINE transcription factor, as underlying cer-x. Comparing the cer-x allelic glossy sheath4.l Bowman Near Isogenic Line BW407 to cv. Bowman revealed an increased cuticular permeability in tissues showing reduced accumulation of β-diketones and altered cuticular metabolic gene expression in BW407. Analyses across the barley pangenome and hundreds of exome-capture datasets revealed high sequence conservation of HvWIN1 and two non-synonymous variants exclusive to the cultivated germplasm. Taken together, we suggest that variation in HvWIN1 controls multiple cuticular features in barley

Photosynthetic antenna size in higher plants is controlled by the plastoquinone redox state at the post-transcriptional rather than transcriptional level.

We analyze the effect of the plastoquinone redox state on the regulation of the light-harvesting antenna size at transcriptional and post-transcriptional levels. This was approached by studying transcription and accumulation of light-harvesting complexes in wild type versus the barley mutant viridis zb63, which is depleted in photosystem I and where plastoquinone is constitutively reduced. We show that the mRNA level of genes encoding antenna proteins is almost unaffected in the mutant; this stability of messenger level is not a peculiarity of antenna-encoding genes, but it extends to all photosynthesis-related genes. In contrast, analysis of protein accumulation by two-dimensional PAGE shows that the mutant undergoes strong reduction of its antenna size, with individual gene products having different levels of accumulation. We conclude that the plastoquinone redox state plays an important role in the long term regulation of chloroplast protein expression. However, its modulation is active at the post-transcriptional rather than transcriptional level

Conserved signalling components coordinate epidermal patterning and cuticle deposition in barley

Faced with terrestrial threats, land plants seal their aerial surfaces with a lipid-rich cuticle. To breathe, plants interrupt their cuticles with adjustable epidermal pores, called stomata, that regulate gas exchange, and develop other specialised epidermal cells such as defensive hairs. Mechanisms coordinating epidermal features remain poorly understood. Addressing this, we studied two loci whose allelic variation causes both cuticular wax-deficiency and misarranged stomata in barley, identifying the underlying genes, Cer-g/ HvYDA1, encoding a YODA-like (YDA) MAPKKK, and Cer-s/ HvBRX-Solo, encoding a single BREVIS-RADIX (BRX) domain protein. Both genes control cuticular integrity, the spacing and identity of epidermal cells, and barley’s distinctive epicuticular wax blooms, as well as stomatal patterning in elevated CO(2) conditions. Genetic analyses revealed epistatic and modifying relationships between HvYDA1 and HvBRX-Solo, intimating that their products participate in interacting pathway(s) linking epidermal patterning with cuticular properties in barley. This may represent a mechanism for coordinating multiple adaptive features of the land plant epidermis in a cultivated cereal

Comparative expression of Cbf genes in the Triticeae under different acclimation induction temperatures

In plants, the C-repeat binding factors (Cbfs) are believed to regulate low-temperature (LT) tolerance. However, most functional studies of Cbfs have focused on characterizing expression after an LT shock and have not quantified differences associated with variable temperature induction or the rate of response to LT treatment. In the Triticeae, rye (Secale cereale L.) is one of the most LT-tolerant species, and is an excellent model to study and compare Cbf LT induction and expression profiles. Here, we report the isolation of rye Cbf genes (ScCbfs) and compare their expression levels in spring- and winter-habit rye cultivars and their orthologs in two winter-habit wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) cultivars. Eleven ScCbfs were isolated spanning all four major phylogenetic groups. Nine of the ScCbfs mapped to 5RL and one to chromosome 2R. Cbf expression levels were variable, with stronger expression in winter- versus spring-habit rye cultivars but no clear relationship with cultivar differences in LT, down-stream cold-regulated gene expression and Cbf expression were detected. Some Cbfs were expressed only at warmer acclimation temperatures in all three species and their expression was repressed at the end of an 8-h dark period at warmer temperatures, which may reflect a temperature-dependent, light-regulated diurnal response. Our work indicates that Cbf expression is regulated by complex genotype by time by induction–temperature interactions, emphasizing that sample timing, induction–temperature and light-related factors must receive greater consideration in future studies involving functional characterization of LT-induced genes in cereals

Allele-Specific HLA Loss and Immune Escape in Lung Cancer Evolution

Immune evasion is a hallmark of cancer. Losing the ability to present neoantigens through human leukocyte antigen (HLA) loss may facilitate immune evasion. However, the polymorphic nature of the locus has precluded accurate HLA copy-number analysis. Here, we present loss of heterozygosity in human leukocyte antigen (LOHHLA), a computational tool to determine HLA allele-specific copy number from sequencing data. Using LOHHLA, we find that HLA LOH occurs in 40% of non-small-cell lung cancers (NSCLCs) and is associated with a high subclonal neoantigen burden, APOBEC-mediated mutagenesis, upregulation of cytolytic activity, and PD-L1 positivity. The focal nature of HLA LOH alterations, their subclonal frequencies, enrichment in metastatic sites, and occurrence as parallel events suggests that HLA LOH is an immune escape mechanism that is subject to strong microenvironmental selection pressures later in tumor evolution. Characterizing HLA LOH with LOHHLA refines neoantigen prediction and may have implications for our understanding of resistance mechanisms and immunotherapeutic approaches targeting neoantigens. Video Abstract [Figure presented] Development of the bioinformatics tool LOHHLA allows precise measurement of allele-specific HLA copy number, improves the accuracy in neoantigen prediction, and uncovers insights into how immune escape contributes to tumor evolution in non-small-cell lung cancer

Characterization of barley albina and xanthia mutants and analysis of gene expression associated with chloroplast development and cold acclimation

I cloroplasti sono la fonte energetica delle cellule vegetali, in grado di catturare la luce e organicare il carbonio tramite il processo fotosintetico. Il cloroplasto ha un ruolo di primaria importanza per il ciclo vitale dei vegetali e quindi lo studio del suo funzionamento e del metabolismo cloroplasto-dipendente \ue8 fondamentale. I plastidi possiedono una propria informazione genetica e gli enzimi necessari per la replicazione del DNA, l\u2019espressione genica e la sintesi proteica. Nonostante ci\uf2 solo una piccola parte delle proteine plastidiali \ue8 codificata dal plastoma (=genoma plastidiale), mentre la maggior parte viene codificata dal genoma nucleare e successivamente importata nel cloroplasto. Il cloroplasto dipende quindi, per il suo corretto funzionamento, anche da geni nucleari. E\u2019 noto, inoltre che le piante, durante il loro ciclo vitale, devono crescere e svilupparsi andando incontro a stress sia biotici che abiotici. E\u2019 quindi ovvio che, nel corso del normale ciclo vitale e al verificarsi di un fattore ambientale, sia necessaria una espressione coordinata dei genomi nucleare e plastidiali al fine di permettere il corretto funzionamento degli organelli e, di conseguenza, dell\u2019intera cellula. Diversi studi hanno enfatizzato l\u2019importanza del cloroplasto durante l\u2019acclimatamento a freddo, sia come fonte di energia, per tutti i cambiamenti richiesti a livello cellulare per l\u2019induzione della resistenza alle basse temperature, sia come possibile fonte di segnali di allarme al resto della cellula, percepiti da fluttuazioni nello stato di ossido/riduzione dell\u2019apparato fotosintetico. Il freddo \ue8 uno dei fattori ambientali che limitano maggiormente la distribuzione geografica e la produttivit\ue0 delle specie vegetali. E\u2019 quindi chiaro che il processo fotosintetico debba interagire con gli altri processi cellulari durante l\u2019esposizione a basse temperature sia fungendo da segnale di allarme, sia integrandosi con le altre vie di trasduzione del segnale che portano all\u2019acclimatamento. Stimoli ambientali e interni confluiscono quindi da diverse vie di trasduzione del segnale modulando l\u2019espressione genica e inducendo le risposte a valle allo scopo di permettere alla pianta di adattarsi al meglio ai cambiamenti ambientali. L\u2019obiettivo di questa tesi \ue8 stato quello di valutare l\u2019influenza del cloroplasto sull\u2019espressione dei geni nucleari e il suo ruolo nel modificare il metabolismo cellulare sia durante le normali condizioni di crescita, sia durante la risposta alle basse temperature. A questo scopo, sono stati analizzati quattro mutanti albina/xantha di orzo che presentano un plastidio bloccato a stadi successivi di sviluppo del cloroplasto. Inoltre \ue8 stato effettuato uno studio su alcuni meccanismi di risposta alle basse temperature indipendenti dal cloroplasto. Un approccio di trascrittomica basato sulla tecnologia microarray, accoppiato con l\u2019utilizzo di mutanti e con uno studio approfondito di alcuni processi metabolici ha portato ad una nuova comprensione dell\u2019effetto del cloroplasto sul genoma nucleare.Chloroplasts are the light-harvesting, carbon-fixing, oxygen-producing energy source of the photosynthetic eukaryotic cells. The chloroplast has a central role for plant life therefore the study of chloroplast functioning and chloroplast dependent metabolism is fundamental. Plastids, having their own genomes, code only for a little part of the chloroplast localized proteins, thus depend, for their functioning, from nuclear codified genes. Moreover higher plants, during their life cycle, have to grow, develop and cope with both abiotic and biotic stress. This means that, during normal growth and as an environmental factor occur, a coordinated expression of nuclear and chloroplast genome is necessary for the assembling, functioning and reprogramming of the organelles and, consequently of the whole cell. Photosynthesis is primarily affected by environmental changes such as light intensity and quality or temperature mediated changes in membrane fluidity. Cold stress is a major environmental factor limiting plants geographical distribution and productivity; exposure to cold in presence of light leads to an excess of adsorbed energy. Photosynthesis itself functions as a sensor of the imbalance between available light and metabolic activity, and regulates the photophysical, photochemical and metabolic process of the chloroplast. It is thus clear that photosynthesis interacts with other processes during cold acclimation involving cross-talk between photosynthetic redox, cold acclimation and sugar signaling pathways to regulate plant acclimation to low temperature. External and internal signals, thus, flow in from various pathways and contribute to modulate gene expression, shaping the downstream response and modifying the plant performance to best exploit environmental conditions. The objective of this thesis was to evaluate the influence of the chloroplast on nuclear gene expression and its role in modifying the cell metabolism either during normal growth condition or during cold acclimation. To this purpose four barley albina/xantha mutants, having plastids blocked at subsequent stages of chloroplast development, were analyzed. Furthermore a study on some cold responsive mechanisms whose expression is independent from the chloroplast was also carried on. The oncoming of whole transcriptome approach, based on array technology, coupled to the use of mutants and accompanied with a deeper investigation of keys metabolic pathways resulted in a new insight of chloroplast effects on nuclear genome

Expression conservation within the circadian clock of a monocot: natural variation at barley <it>Ppd-H1</it> affects circadian expression of flowering time genes, but not clock orthologs

Abstract Background The circadian clock is an endogenous mechanism that coordinates biological processes with daily changes in the environment. In plants, circadian rhythms contribute to both agricultural productivity and evolutionary fitness. In barley, the photoperiod response regulator and flowering-time gene Ppd-H1 is orthologous to the Arabidopsis core-clock gene PRR7. However, relatively little is known about the role of Ppd-H1 and other components of the circadian clock in temperate crop species. In this study, we identified barley clock orthologs and tested the effects of natural genetic variation at Ppd-H1 on diurnal and circadian expression of clock and output genes from the photoperiod-response pathway. Results Barley clock orthologs HvCCA1, HvGI, HvPRR1, HvPRR37 (Ppd-H1), HvPRR73, HvPRR59 and HvPRR95 showed a high level of sequence similarity and conservation of diurnal and circadian expression patterns, when compared to Arabidopsis. The natural mutation at Ppd-H1 did not affect diurnal or circadian cycling of barley clock genes. However, the Ppd-H1 mutant was found to be arrhythmic under free-running conditions for the photoperiod-response genes HvCO1, HvCO2, and the MADS-box transcription factor and vernalization responsive gene Vrn-H1. Conclusion We suggest that the described eudicot clock is largely conserved in the monocot barley. However, genetic differentiation within gene families and differences in the function of Ppd-H1 suggest evolutionary modification in the angiosperm clock. Our data indicates that natural variation at Ppd-H1 does not affect the expression level of clock genes, but controls photoperiodic output genes. Circadian control of Vrn-H1 in barley suggests that this vernalization responsive gene is also controlled by the photoperiod-response pathway. Structural and functional characterization of the barley circadian clock will set the basis for future studies of the adaptive significance of the circadian clock in Triticeae species.</p

Transcriptome Analysis of Cold Acclimation in Barley Albina and Xantha Mutants

Previously, we have shown that barley (Hordeum vulgare) plants carrying a mutation preventing chloroplast development are completely frost susceptible as well as impaired in the expression of several cold-regulated genes. Here we investigated the transcriptome of barley albina and xantha mutants and the corresponding wild type to assess the effect of the chloroplast on expression of cold-regulated genes. First, by comparing control wild type against cold-hardened wild-type plants 2,735 probe sets with statistically significant changes (P = 0.05; ≥2-fold change) were identified. Expression of these wild-type cold-regulated genes was then analyzed in control and cold-hardened mutants. Only about 11% of the genes cold regulated in wild type were regulated to a similar extent in all genotypes (chloroplast-independent cold-regulated genes); this class includes many genes known to be under C-repeat binding factor control. C-repeat binding factor genes were also equally induced in mutants and wild-type plants. About 67% of wild-type cold-regulated genes were not regulated by cold in any mutant (chloroplast-dependent cold-regulated genes). We found that the lack of cold regulation in the mutants is due to the presence of signaling pathway(s) normally cold activated in wild type but constitutively active in the mutants, as well as to the disruption of low-temperature signaling pathway(s) due to the absence of active chloroplasts. We also found that photooxidative stress signaling pathway is constitutively active in the mutants. These results demonstrate the major role of the chloroplast in the control of the molecular adaptation to cold