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In the realm of agricultural sustainability, the utilization of plant genetic resources for enhanced disease resistance is paramount. Preservation efforts in genebanks are justified by their potential contributions to future crop improvement. To capitalize on the potential of plant genetic resources, we focused on a barley core collection from the German ex situ genebank and contrasted it with a European elite collection. The phenotypic assessment included 812 plant genetic resources and 298 elites, with a particular emphasis on four disease traits (Puccinia hordei, Blumeria graminis hordei, Ramularia collo-cygni, and Rhynchosporium commune). An integrated genome-wide association study, employing both Bayesian-information and linkage-disequilibrium iteratively nested keyway (BLINK) and a linear mixed model, was performed to unravel the genetic underpinnings of disease resistance. A total of 932 marker-trait associations were identified and assigned to 49 quantitative trait loci. The accumulation of novel and rare resistance alleles significantly bolstered the overall resistance level in plant genetic resources. Three plant genetic resources donors with high counts of novel/rare alleles and exhibiting exceptional resistance to leaf rust and powdery mildew were identified, offering promise for targeted pre-breeding goals and enhanced resilience in future varieties. Our findings underscore the critical contribution of plant genetic resources to strengthening crop resilience and advancing sustainable agricultural practices., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Optimizing photosynthesis is considered an important strategy for improving crop yields to ensure food security. To evaluate the potential of using photosynthesis-related parameters in crop breeding programs, we measured chlorophyll fluorescence along with growth-related and morphological traits of 23 barley inbred lines across different developmental stages in field conditions. The photosynthesis-related parameters were highly variable, changing with light intensity and developmental progression of plants. Yet, the variation in photosystem II quantum yield observed among the inbred lines in the field largely reflected the variation in CO2 assimilation properties in controlled climate chamber conditions, confirming that the chlorophyll fluorescence-based technique can provide proxy parameters of photosynthesis to explore genetic variation under field conditions. Heritability (H2) of the photosynthesis-related parameters in the field ranged from 0.16 for the quantum yield of non-photochemical quenching to 0.78 for the fraction of open photosystem II center. Two parameters, the maximum photosystem II efficiency in the light-adapted state (H2=0.58) and the total non-photochemical quenching (H2=0.53), showed significant positive and negative correlations, respectively, with yield-related traits (dry weight per plant and net straw weight) in the barley inbred lines. These results indicate the possibility of improving crop yield through optimizing photosynthetic light use efficiency by conventional breeding programs., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Partial resistance to multiple biotrophic fungal pathogens in wheat (Triticum aestivum L.) is conferred by a variant of the Lr67 gene, which encodes a hexose-proton symporter. Two mutations (G144R and V387L) differentiate the resistant and susceptible protein variants (Lr67res and Lr67sus). Lr67res lacks sugar transport capability and was associated with anion transporter-like properties when expressed in Xenopus laevis oocytes. Here, we extended this functional characterization to include yeast and in planta studies. The Lr67res allele, but not Lr67sus, induced sensitivity to ions in yeast (including NaCl, LiCl, and KI), which is consistent with our previous observations that Lr67res expression in oocytes induces novel ion fluxes. We demonstrate that another naturally occurring single amino acid variant in wheat, containing only the Lr67G144R mutation, confers rust resistance. Transgenic barley plants expressing the orthologous HvSTP13 gene carrying the G144R and V387L mutations were also more resistant to Puccinia hordei infection. NaCl treatment of pot-grown adult wheat plants with the Lr67res allele induced leaf tip necrosis and partial leaf rust resistance. An Lr67res-like function can be introduced into orthologous plant hexose transporters via single amino acid mutation, highlighting the strong possibility of generating disease resistance in other crops, especially with gene editing., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

The HD-ZIP class I transcription factor Homeobox 1 (HvHOX1), also known as Vulgare Row-type Spike 1 (VRS1) or Six-rowed Spike 1, regulates lateral spikelet fertility in barley (Hordeum vulgare L.). It was shown that HvHOX1 has a high expression only in lateral spikelets, while its paralog HvHOX2 was found to be expressed in different plant organs. Yet, the mechanistic functions of HvHOX1 and HvHOX2 during spikelet development are still fragmentary. Here, we show that compared with HvHOX1, HvHOX2 is more highly conserved across different barley genotypes and Hordeum species, hinting at a possibly vital but still unclarified biological role. Using bimolecular fluorescence complementation, DNA-binding, and transactivation assays, we validate that HvHOX1 and HvHOX2 are bona fide transcriptional activators that may potentially heterodimerize. Accordingly, both genes exhibit similar spatiotemporal expression patterns during spike development and growth, albeit their mRNA levels differ quantitatively. We show that HvHOX1 delays the lateral spikelet meristem differentiation and affects fertility by aborting the reproductive organs. Interestingly, the ancestral relationship of the two genes inferred from their co-expressed gene networks suggested that HvHOX1 and HvHOX2 might play a similar role during barley spikelet development. However, CRISPR-derived mutants of HvHOX1 and HvHOX2 demonstrated the suppressive role of HvHOX1 on lateral spikelets, while the loss of HvHOX2 does not influence spikelet development. Collectively, our study shows that through the suppression of reproductive organs, lateral spikelet fertility is regulated by HvHOX1, whereas HvHOX2 is dispensable for spikelet development in barley., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Barley is a staple crop of major global importance and relatively resilient to a wide range of stress factors in the field. Transgenic reporter lines to investigate physiological parameters during stress treatments remain scarce. We generated and characterized transgenic homozygous barley lines (cv. Golden Promise Fast) expressing the genetically encoded biosensor Grx1-roGFP2, which indicates the redox potential of the major antioxidant glutathione in the cytosol. Our results demonstrated functionality of the sensor in living barley plants. We determined the glutathione redox potential (EGSH) of the cytosol to be in the range of -308 mV to -320 mV. EGSH was robust against a combined NaCl (150 mM) and water deficit treatment (-0.8 MPa) but responded with oxidation to infiltration with the phytotoxic secretome of the necrotrophic fungus Botrytis cinerea. The generated reporter lines are a novel resource to study biotic and abiotic stress resilience in barley, pinpointing that even severe abiotic stress leading to a growth delay does not automatically induce cytosolic EGSH oxidation, while necrotrophic pathogens can undermine this robustness., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Flowering time and plant height are two critical determinants of yield potential in barley (Hordeum vulgare). Despite their role in plant physiological regulation, a complete overview of the genetic complexity of flowering time and plant height regulation in barley is still lacking. Using a double round-robin population originated from the crossings of 23 diverse parental inbred lines, we aimed to determine the variance components in the regulation of flowering time and plant height in barley as well as to identify new genetic variants by single and multi-population QTL analyses and allele mining. Despite similar genotypic variance, we observed higher environmental variance components for plant height than flowering time. Furthermore, we detected new QTLs for flowering time and plant height. Finally, we identified a new functional allelic variant of the main regulatory gene Ppd-H1. Our results show that the genetic architecture of flowering time and plant height might be more complex than reported earlier and that a number of undetected, small effect, or low-frequency genetic variants underlie the control of these two traits., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

One of the challenges for global food security is to reliably and sustainably improve the grain yield of cereal crops. One solution is to modify the architecture of the grain-bearing inflorescence to optimize for grain number and size. Cereal inflorescences are complex structures, with determinacy, branching patterns, and spikelet/floret growth patterns that vary by species. Recent decades have witnessed rapid advancements in our understanding of the genetic regulation of inflorescence architecture in rice, maize, wheat, and barley. Here, we summarize current knowledge on key genetic factors underlying the different inflorescence morphologies of these crops and model plants (Arabidopsis and tomato), focusing particularly on the regulation of inflorescence meristem determinacy and spikelet meristem identity and determinacy. We also discuss strategies to identify and utilize these superior alleles to optimize inflorescence architecture and, ultimately, improve crop grain yield., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Barley is a long-day plant with a major gene (PPD-H1) that determines its photoperiod sensitivity. Under long days (i.e. 16 h), flowering occurs earlier in sensitive (Ppd-H1) than in insensitive (ppd-H1) genotypes, while under short days (i.e. 12 h) both flower late and more or less simultaneously. We hypothesized that (i) the sensitive line should flower later than the insensitive line under very short days (<12 h), and (ii) both the sensitive and insensitive lines should have similar phenology under very long days (>18 h). When comparing a pair of spring isogenic lines for sensitive and insensitive PPD-H1 alleles (introgressing the PPD-H1 allele into the barley cultivar 'WI4441'), we found responses fully in line with expectations for the commonly explored range from 12 to 16-18 h. When the responses were extended to very short days, sensitivity increased noticeably, and time to flowering of the sensitive line was longer than that of the insensitive one. Under very long days, the sensitive line did not respond further (it seemed to have reached its minimum time to flowering under a 16 h period), while the insensitive line continued shortening its time to flowering until c. 21 h. Consequently, both lines flowered similarly under very long days, which opens opportunities to easily test for differences in earliness per se, as in wheat., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Plants often face simultaneous abiotic and biotic stress conditions; however, physiological and transcriptional responses under such combined stress conditions are still not fully understood. Spring barley (Hordeum vulgare) is susceptible to Fusarium head blight (FHB), which is strongly affected by weather conditions. We therefore studied the potential influence of drought on FHB severity and plant responses in three varieties of different susceptibility. We found strongly reduced FHB severity in susceptible varieties under drought. The number of differentially expressed genes (DEGs) and strength of transcriptomic regulation reflected the concentrations of physiological stress markers such as abscisic acid or fungal DNA contents. Infection-related gene expression was associated with susceptibility rather than resistance. Weighted gene co-expression network analysis revealed 18 modules of co-expressed genes that reflected the pathogen- or drought-response in the three varieties. A generally infection-related module contained co-expressed genes for defence, programmed cell death, and mycotoxin detoxification, indicating that the diverse genotypes used a similar defence strategy towards FHB, albeit with different degrees of success. Further, DEGs showed co-expression in drought- or genotype-associated modules that correlated with measured phytohormones or the osmolyte proline. The combination of drought stress with infection led to the highest numbers of DEGs and resulted in a modular composition of the single-stress responses rather than a specific transcriptional output., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Correct floral development is the result of a sophisticated balance of molecular cues. Floral mutants provide insight into the main genetic determinants that integrate these cues, as well as providing opportunities to assess functional variation across species. In this study, we characterize the barley (Hordeum vulgare) multiovary mutants mov2.g and mov1, and propose causative gene sequences: a C2H2 zinc-finger gene HvSL1 and a B-class gene HvMADS16, respectively. In the absence of HvSL1, florets lack stamens but exhibit functional supernumerary carpels, resulting in multiple grains per floret. Deletion of HvMADS16 in mov1 causes homeotic conversion of lodicules and stamens into bract-like organs and carpels that contain non-functional ovules. Based on developmental, genetic, and molecular data, we propose a model by which stamen specification in barley is defined by HvSL1 acting upstream of HvMADS16. The present work identifies strong conservation of stamen formation pathways with other cereals, but also reveals intriguing species-specific differences. The findings lay the foundation for a better understanding of floral architecture in Triticeae, a key target for crop improvement., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

The description of long photoperiod sensitivity in wheat and barley is a cause of confusion for researchers working with these crops, usually accustomed to free exchange of physiological and genetic knowledge of such similar crops. Indeed, wheat and barley scientists customarily quote studies of either crop species when researching one of them. Among their numerous similarities, the main gene controlling the long photoperiod sensitivity is the same in both crops (PPD1; PPD-H1 in barley and PPD-D1 in hexaploid wheat). However, the photoperiod responses are different: (i) the main dominant allele inducing shorter time to anthesis is the insensitive allele in wheat (Ppd-D1a) but the sensitive allele in barley (Ppd-H1) (i.e. sensitivity to photoperiod produces opposite effects on time to heading in wheat and barley); (ii) the main 'insensitive' allele in wheat, Ppd-D1a, does confer insensitivity, whilst that of barley reduces the sensitivity but still responds to photoperiod. The different behaviour of PPD1 genes in wheat and barley is put in a common framework based on the similarities and differences of the molecular bases of their mutations, which include polymorphism at gene expression levels, copy number variation, and sequence of coding regions. This common perspective sheds light on a source of confusion for cereal researchers, and prompts us to recommend accounting for the photoperiod sensitivity status of the plant materials when conducting research on genetic control of phenology. Finally, we provide advice to facilitate the management of natural PPD1 diversity in breeding programmes and suggest targets for further modification through gene editing, based on mutual knowledge on the two crops., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Global climate change has dramatically increased flooding events, which have a strong impact on crop production. Barley (Hordeum vulgare) is one of the most important cereals and its cultivation includes a broad range of different environments. We tested the capacity to germinate of a large barley panel after a short period of submergence followed by a period of recovery. We demonstrate that sensitive barley varieties activate underwater secondary dormancy because of a lower permeability to oxygen dissolved in water. In sensitive barley accessions, secondary dormancy is removed by nitric oxide donors. The results of a genome-wide association study uncovered a Laccase gene located in a region of significant marker-trait association that is differently regulated during grain development and plays a key role in this process. Our findings will help breeders to improve the genetics of barley, thereby increasing the capacity of seeds to germinate after a short period of flooding., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

EARLY FLOWERING 3 (ELF3) is an important regulator of various physiological and developmental processes and hence may serve to improve plant adaptation which will be essential for future plant breeding. To expand the limited knowledge on barley ELF3 in determining agronomic traits, we conducted field studies with heterogeneous inbred families (HIFs) derived from selected lines of the wild barley nested association mapping population HEB-25. During two growing seasons, phenotypes of nearly isogenic HIF sister lines, segregating for exotic and cultivated alleles at the ELF3 locus, were compared for 10 developmental and yield-related traits. We determine novel exotic ELF3 alleles and show that HIF lines, carrying the exotic ELF3 allele, accelerated plant development compared with the cultivated ELF3 allele, depending on the genetic background. Remarkably, the most extreme effects on phenology could be attributed to one exotic ELF3 allele differing from the cultivated Barke ELF3 allele in only one single nucleotide polymorphism (SNP). This SNP causes an amino acid substitution (W669G), which as predicted has an impact on the protein structure of ELF3. Consequently, it may affect phase separation behaviour and nano-compartment formation of ELF3 and, potentially, also its local cellular interactions causing significant trait differences between HIF sister lines., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Defense responses in plants are based on complex biochemical processes. Systemic acquired resistance (SAR) helps to fight infections by (hemi-)biotrophic pathogens. One important signaling molecule in SAR is pipecolic acid (Pip), accumulation of which is dependent on the aminotransferase ALD1 in Arabidopsis. While exogenous Pip primes defense responses in the monocotyledonous cereal crop barley (Hordeum vulgare), it is currently unclear if endogenous Pip plays a role in disease resistance in monocots. Here, we generated barley ald1 mutants using CRISPR/Cas9, and assessed their capacity to mount SAR. Endogenous Pip levels were reduced after infection of the ald1 mutant, and this altered systemic defense against the fungus Blumeria graminis f. sp. hordei. Furthermore, Hvald1 plants did not emit nonanal, one of the key volatile compounds that are normally emitted by barley plants after the activation of SAR. This resulted in the inability of neighboring plants to perceive and/or respond to airborne cues and prepare for an upcoming infection, although HvALD1 was not required in the receiver plants to mediate the response. Our results highlight the crucial role of endogenous HvALD1 and Pip for SAR, and associate Pip, in particular together with nonanal, with plant-to-plant defense propagation in the monocot crop barley., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Rabl organization is a type of interphase chromosome arrangement with centromeres and telomeres clustering at opposite nuclear poles. Here, we analyzed nuclear morphology and chromosome organization in cycling and endoreduplicated nuclei isolated from embryo and endosperm tissues of developing barley seeds. We show that endoreduplicated nuclei have an irregular shape, less sister chromatid cohesion at 5S rDNA loci, and a reduced amount of centromeric histone CENH3. While the chromosomes of the embryo and endosperm nuclei are initially organized in Rabl configuration, the centromeres and telomeres are intermingled within the nuclear space in the endoreduplicated nuclei with an increasing endoreduplication level. Such a loss of chromosome organization suggests that Rabl configuration is introduced and further reinforced by mitotic divisions in barley cell nuclei in a tissue- and seed age-dependent manner., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

To counter projected reductions in yields of the major crop barley, it is essential to elucidate the mechanisms of its resilience. To assist such efforts, we collected grains from plants grown in fields at 12 testing stations, with suitable temperature and precipitation gradients for identifying environmentally induced changes in their protein and metabolite contents. We then subjected the grains to detailed molecular analysis. The results showed that numerous metabolites and at least a quarter of the grain protein content was modulated by the environment, and provided insights into barley seed production under abiotic stress, including alterations in ribosomal proteins, heatshock protein 70 family proteins, inhibitors, storage proteins, and lipid droplet formation. Potential positive and negative markers of yield were also identified, including the phenolic compound catechin and storage protein levels, respectively. Complementary analyses of barley seedlings and Arabidopsis seeds, respectively, confirmed the role of the identified proteins in abiotic stress responses and highlighted evolutionarily conserved mechanisms. In addition, accelerated ageing experiments revealed that variations in the environment had stronger effects on seed longevity than the genotype. Finally, seeds with the highest longevity differed from the others in gibberellin contents, H2O2 metabolism, and levels of >250 proteins, providing novel targets for improving resilience., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Encasements formed around haustoria and biotrophic hyphae as well as hypersensitive reaction (HR) cell death are essential plant immune responses to filamentous pathogens. In this study we examine the components that may contribute to the absence of these responses in susceptible barley attacked by the powdery mildew fungus. We find that the effector CSEP0162 from this pathogen targets plant MONENSIN SENSITIVITY1 (MON1), which is important for the fusion of multivesicular bodies to their target membranes. Overexpression of CSEP0162 and silencing of barley MON1 both inhibit encasement formation. We find that the Arabidopsis ecotype No-0 has resistance to powdery mildew, and that this is partially dependent on MON1. Surprisingly, we find the MON1-dependent resistance in No-0 not only includes an encasement response, but also an effective HR. Similarly, silencing of MON1 in barley also blocks Mla3-mediated HR-based powdery mildew resistance. Our results indicate that MON1 is a vital plant immunity component, and we speculate that the barley powdery mildew fungus introduces the effector CSEP0162 to target MON1 and hence reduce encasement formation and HR., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Grain number, size and weight primarily determine the yield of barley. Although the genes regulating grain number are well studied in barley, the genetic loci and the causal gene for sink capacity are poorly understood. Therefore, the primary objective of our work was to dissect the genetic architecture of grain size and weight in barley. We used a multi-parent population developed from a genetic cross between 23 diverse barley inbreds in a double round-robin design. Seed size-related parameters such as grain length, grain width, grain area and thousand-grain weight were evaluated in the HvDRR population comprising 45 recombinant inbred line sub-populations. We found significant genotypic variation for all seed size characteristics, and observed 84% or higher heritability across four environments. The quantitative trait locus (QTL) detection results indicate that the genetic architecture of grain size is more complex than previously reported. In addition, both cultivars and landraces contributed positive alleles at grain size QTLs. Candidate genes identified using genome-wide variant calling data for all parental inbred lines indicated overlapping and potential novel regulators of grain size in cereals. Furthermore, our results indicated that sink capacity was the primary determinant of grain weight in barley., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Leaf senescence is a developmental process allowing nutrient remobilization to sink organs. We characterized flag leaf senescence at 7, 14, and 21 d past anthesis in two near-isogenic barley lines varying in the allelic state of the HvNAM1 transcription factor gene, which influences senescence timing. Metabolomics and microscopy indicated that, as senescence progressed, thylakoid lipids were transiently converted to neutral lipids accumulating in lipid droplets. Senescing leaves also exhibited an accumulation of sugars including glucose, while nitrogen compounds (nucleobases, nucleotides, and amino acids) decreased. RNA-Seq analysis suggested lipid catabolism via β-oxidation and the glyoxylate cycle, producing carbon skeletons and feeding respiration as a replacement of the diminished carbon supply from photosynthesis. Comparison of the two barley lines highlighted a more prominent up-regulation of heat stress transcription factor- and chaperone-encoding genes in the late-senescing line, suggesting a role for these genes in the control of leaf longevity. While numerous genes with putative roles in nitrogen remobilization were up-regulated in both lines, several peptidases, nucleases, and nitrogen transporters were more highly induced in the early-senescing line; this finding identifies processes and specific candidates which may affect nitrogen remobilization from senescing barley leaves, downstream of the HvNAM1 transcription factor., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Recent work has identified several proteins involved in starch granule initiation, the first step of starch synthesis. However, the degree of conservation in the granule initiation process remains poorly understood, especially among grass species differing in patterns of carbohydrate turnover in leaves, and granule morphology in the endosperm. We therefore compared mutant phenotypes of Hordeum vulgare (barley), Triticum turgidum (durum wheat), and Brachypodium distachyon defective in PROTEIN TARGETING TO STARCH 2 (PTST2), a key granule initiation protein. We report striking differences across species and organs. Loss of PTST2 from leaves resulted in fewer, larger starch granules per chloroplast and normal starch content in wheat, fewer granules per chloroplast and lower starch content in barley, and almost complete loss of starch in Brachypodium. The loss of starch in Brachypodium leaves was accompanied by high levels of ADP-glucose and detrimental effects on growth and physiology. Additionally, we found that loss of PTST2 increased granule initiation in Brachypodium amyloplasts, resulting in abnormal compound granule formation throughout the seed. These findings suggest that the importance of PTST2 varies greatly with the genetic and developmental background and inform the extent to which the gene can be targeted to improve starch in crops., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Crops are exposed to myriad abiotic and biotic stressors with negative consequences. Two stressors that are expected to increase under climate change are drought and infestation with herbivorous insects, including important aphid species. Expanding our understanding of the impact drought has on the plant-aphid relationship will become increasingly important under future climate scenarios. Here we use a previously characterized plant-aphid system comprising a susceptible variety of barley, a wild relative of barley with partial aphid resistance, and the bird cherry-oat aphid to examine the drought-plant-aphid relationship. We show that drought has a negative effect on plant physiology and aphid fitness, and provide evidence to suggest that plant resistance influences aphid responses to drought stress. Furthermore, we show that the expression of thionin genes, plant defensive compounds that contribute to aphid resistance, increase in susceptible plants exposed to drought stress but remain at constant levels in the partially resistant plant, suggesting that they play an important role in determining the success of aphid populations. This study highlights the role of plant defensive processes in mediating the interactions between the environment, plants, and herbivorous insects., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Tiller formation is a key agronomic determinant for grain yield in cereal crops. The modulation of this trait is controlled by transcriptional regulators and plant hormones, tightly regulated by external environmental conditions. While endogenous (genetic) and exogenous (environmental factors) triggers for tiller formation have mostly been investigated separately, it has remained elusive how they are integrated into the developmental program of this trait. The transcription factor gene INTERMEDIUM-C (INT-C), which is the barley ortholog of the maize domestication gene TEOSINTE BRANCHED1 (TB1), has a prominent role in regulating tiller bud outgrowth. Here we show that INT-C is expressed in tiller buds, required for bud growth arrest in response to shade. In contrast to wild-type plants, int-c mutant plants are impaired in their shade response and do not stop tiller production after shading. Gene expression levels of INT-C are up-regulated under light-limiting growth conditions, and down-regulated after decapitation. Transcriptome analysis of wild-type and int-c buds under control and shading conditions identified target genes of INT-C that belong to auxin and gibberellin biosynthesis and signaling pathways. Our study identifies INT-C as an integrator of the shade response into tiller formation, which is prerequisite for implementing shading responses in the breeding of cereal crops., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

The potential to increase barley grain yield lies in the indeterminate nature of its inflorescence meristem, which produces spikelets, the basic reproductive unit in grasses that are linked to reproductive success. During early reproductive growth, barley spikes pass through the maximum yield potential-a stage after which no new spikelet ridges are produced. Subsequently, spikelet abortion (SA), a phenomenon in which spikelets abort during spike growth, imposes a bottleneck for increasing the grain yield potential. Here, we studied the potential of main culm spikes by counting potential spikelet number (PSN) and final spikelet number (FSN), and computed the corresponding SA (%) in a panel of 417 six-rowed spring barleys. Our phenotypic data analyses showed a significantly large within- and across-years genotypic variation with high broad-sense heritability estimates for all the investigated traits, including SA. Asian accessions displayed the lowest SA, indicating the presence of favourable alleles that may be exploited in breeding programs. A significantly negative Pearson's product-moment correlation was observed between FSN and SA. Our path analysis revealed that PSN and FSN explain 93% of the observed phenotypic variability for SA, with PSN behaving as a suppressor trait that magnifies the effect of FSN. Based on a large set of diverse barley accessions, our results provide a deeper phenotypic understanding of the quantitative genetic nature of SA, its association with traits of high agronomic importance, and a resource for further genetic analyses., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Flowering plants reproduce sexually by combining a haploid male and female gametophyte during fertilization. Male gametophytes are localized in the anthers, each containing reproductive (meiocyte) and non-reproductive tissue necessary for anther development and maturation. Meiosis, where chromosomes pair and exchange their genetic material during a process called recombination, is one of the most important and sensitive stages in breeding, ensuring genetic diversity. Most anther development studies have focused on transcript variation, but very few have been correlated with protein abundance. Taking advantage of a recently published barley anther transcriptomic (BAnTr) dataset and a newly developed sensitive mass spectrometry-based approach to analyse the barley anther proteome, we conducted high-resolution mass spectrometry analysis of barley anthers, collected at six time points and representing their development from pre-meiosis to metaphase. Each time point was carefully staged using immunocytology, providing a robust and accurate staging mirroring our previous BAnTr dataset. We identified >6100 non-redundant proteins including 82 known and putative meiotic proteins. Although the protein abundance was relatively stable throughout prophase I, we were able to quantify the dynamic variation of 336 proteins. We present the first quantitative comparative proteomics study of barley anther development during meiotic prophase I when the important process of homologous recombination is taking place., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Plants have evolved complex mechanisms to adapt to the changing nitrogen levels in the environment. In Arabidopsis, more than a dozen nitrate signaling regulatory genes have been characterized, including the NODULE INCEPTION-LIKE PROTEIN (AtNLP) genes, which play essential roles in nitrate signaling. However, whether NLP genes in the Triticeae crops are involved in nitrate regulation and nitrogen use efficiency (NUE) remains unknown. Here, we isolated a barley (Hordeum vulgare L.) mutant, hvnlp2-1, from a TILLING (Targeting Local Lesions IN Genomes) population and constructed two RNAi lines, hvnlp2-2 and hvnlp2-3, to study the function of HvNLP2. The expression of the nitrate-responsive genes was substantially inhibited after nitrate treatment in the hvnlp2 mutants, indicating that HvNLP2 controls nitrate signaling. Nitrate content was significantly higher in the hvnlp2 mutants, which may result from the decreased assimilation of nitrogen caused by reduced nitrate reductase activity and expression of nitrate assimilatory genes. HvNLP2 is localized to the nucleus in the presence of nitrate. Further investigation showed that HvNLP2 binds to and activates the nitrate-responsive cis-elements. Moreover, hvnlp2 exhibited reduced biomass, seed yield, and NUE. Therefore, HvNLP2 controls nitrate signaling and plays an important role in NUE., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Plants activate biochemical responses to combat stress. (Hemi-)biotrophic pathogens are fended off by systemic acquired resistance (SAR), a primed state allowing plants to respond faster and more strongly upon subsequent infection. Here, we show that SAR-like defences in barley (Hordeum vulgare) are propagated between neighbouring plants, which respond with enhanced resistance to the volatile cues from infected senders. The emissions of the sender plants contained 15 volatile organic compounds (VOCs) associated with infection. Two of these, β-ionone and nonanal, elicited resistance upon plant exposure. Whole-genome transcriptomics analysis confirmed that interplant propagation of defence in barley is established as a form of priming. Although gene expression changes were more pronounced after challenge infection of the receiver plants with Blumeria graminis f. sp. hordei, differential gene expression in response to the volatile cues of the sender plants included an induction of HISTONE DEACETYLASE 2 (HvHDA2) and priming of TETRATRICOPEPTIDE REPEAT-LIKE superfamily protein (HvTPL). Because HvHDA2 and HvTPL transcript accumulation was also enhanced by exposure of barley to β-ionone and nonanal, our data identify both genes as possible defence/priming markers in barley. Our results suggest that VOCs and plant-plant interactions are relevant for possible crop protection strategies priming defence responses in barley., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Determining the grain yield potential contributed by grain number is a step towards advancing the yield of cereal crops. To achieve this aim, it is pivotal to recognize the maximum yield potential (MYP) of the crop. In barley (Hordeum vulgare L.), the MYP is defined as the maximum spikelet primordia number of a spike. Many barley studies assumed the awn primordium (AP) stage to be the MYP stage regardless of genotypes and growth conditions. From our spikelet-tracking experiments using the two-rowed cultivar Bowman, we found that the MYP stage can be different from the AP stage. Importantly, we find that the occurrence of inflorescence meristem deformation and its loss of activity coincided with the MYP stage, indicating the end of further spikelet initiation. Thus, we recommend validating the barley MYP stage with the shape of the inflorescence meristem and propose this approach (named 'spikelet stop') for MYP staging. To clarify the relevance of AP and MYP stages, we compared the MYP stage and the MYP in 27 barley accessions (two- and six-rowed accessions) grown in the greenhouse and in the field. Our results reveal that the MYP stage can be reached at various developmental stages, which greatly depend on the genotype and growth conditions. Furthermore, we propose that the MYP stage and the time to reach the MYP stage can be used to determine yield potential in barley. Based on our findings, we suggest key steps for the identification of the MYP stage in barley that may also be applied in a related crop such as wheat., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Gaining knowledge on fundamental interactions of various yield components is crucial to improve yield potential in small grain cereals. It is well known in barley that increasing grain number greatly improves yield potential; however, the yield components determining grain number and their association in barley row types are less explored. In this study, we assessed different yield components such as potential spikelet number (PSN), spikelet survival (SSL), spikelet number (SN), grain set (GS), and grain survival (GSL), as well as their interactions with grain number by using a selected panel of two- and six-rowed barley types. Also, to analyze the stability of these interactions, we performed the study in the greenhouse and the field. From this study, we found that in two-rowed barley, grain number determination is strongly influenced by PSN rather than SSL and/or GS in both growth conditions. Conversely, in six-rowed barley, grain number is associated with SSL instead of PSN and/or GS. Thus, our study showed that increasing grain number might be possible by augmenting PSN in two-rowed genotypes, while for six-rowed genotypes SSL needs to be improved. We speculate that this disparity of grain number determination in barley row types might be due to the fertility of lateral spikelets. Collectively, this study revealed that grain number in two-rowed barley largely depends on the developmental trait, PSN, while in six-rowed barley, it mainly follows the ability for SSL., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

During barley germination, cysteine proteases are essential in the mobilization of storage compounds providing peptides and amino acids to sustain embryo growth until photosynthesis is completely established. Knockdown barley plants, generated by artificial miRNA, for the cathepsins B- and F-like HvPap-19 and HvPap-1 genes, respectively, showed less cysteine protease activities and consequently lower protein degradation. The functional redundancy between proteases triggered an enzymatic compensation associated with an increase in serine protease activities in both knockdown lines, which was not sufficient to maintain germination rates and behaviour. Concomitantly, these transgenic lines showed alterations in the accumulation of protein and carbohydrates in the grain. While the total amount of protein increased in both transgenic lines, the starch content decreased in HvPap-1 knockdown lines and the sucrose concentration was reduced in silenced HvPap-19 grains. Consequently, phenotypes of HvPap-1 and HvPap-19 artificial miRNA lines showed a delay in the grain germination process. These data demonstrate the potential of exploring the properties of barley proteases for selective modification and use in brewing or in the livestock feeding industry., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

We profiled the grain oligosaccharide content of 154 two-row spring barley genotypes and quantified 27 compounds, mainly inulin- and neoseries-type fructans, showing differential abundance. Clustering revealed two profile groups where the 'high' set contained greater amounts of sugar monomers, sucrose, and overall fructans, but lower fructosylraffinose. A genome-wide association study (GWAS) identified a significant association for the variability of two fructan types: neoseries-DP7 and inulin-DP9, which showed increased strength when applying a novel compound ratio-GWAS approach. Gene models within this region included three known fructan biosynthesis genes (fructan:fructan 1-fructosyltransferase, sucrose:sucrose 1-fructosyltransferase, and sucrose:fructan 6-fructosyltransferase). Two other genes in this region, 6(G)-fructosyltransferase and vacuolar invertase1, have not previously been linked to fructan biosynthesis and showed expression patterns distinct from those of the other three genes, including exclusive expression of 6(G)-fructosyltransferase in outer grain tissues at the storage phase. From exome capture data, several single nucleotide polymorphisms related to inulin- and neoseries-type fructan variability were identified in fructan:fructan 1-fructosyltransferase and 6(G)-fructosyltransferase genes. Co-expression analyses uncovered potential regulators of fructan biosynthesis including transcription factors. Our results provide the first scientific evidence for the distinct biosynthesis of neoseries-type fructans during barley grain maturation and reveal novel gene candidates likely to be involved in the differential biosynthesis of various types of fructan in barley., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Seeds are complex biological systems comprising three genetically distinct tissues: embryo, endosperm, and maternal tissues (including seed coats and pericarp) nested inside one another. Cereal grains represent a special type of seeds, with the largest part formed by the endosperm, a specialized triploid tissue ensuring embryo protection and nourishment. We investigated dynamic changes in DNA content in three of the major seed tissues from the time of pollination up to the dry seed. We show that the cell cycle is under strict developmental control in different seed compartments. After an initial wave of active cell division, cells switch to endocycle and most endoreduplication events are observed in the endosperm and seed maternal tissues. Using different barley cultivars, we show that there is natural variation in the kinetics of this process. During the terminal stages of seed development, specific and selective loss of endoreduplicated nuclei occurs in the endosperm. This is accompanied by reduced stability of the nuclear genome, progressive loss of cell viability, and finally programmed cell death. In summary, our study shows that endopolyploidization and cell death are linked phenomena that frame barley grain development., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Drought impairs growth and spike development, and is therefore a major cause of yield losses in the temperate cereals barley and wheat. Here, we show that the photoperiod response gene PHOTOPERIOD-H1 (Ppd-H1) interacts with drought stress signals to modulate spike development. We tested the effects of a continuous mild and a transient severe drought stress on developmental timing and spike development in spring barley cultivars with a natural mutation in ppd-H1 and derived introgression lines carrying the wild-type Ppd-H1 allele from wild barley. Mild drought reduced the spikelet number and delayed floral development in spring cultivars but not in the introgression lines with a wild-type Ppd-H1 allele. Similarly, drought-triggered reductions in plant height, and tiller and spike number were more pronounced in the parental lines compared with the introgression lines. Transient severe stress halted growth and floral development; upon rewatering, introgression lines, but not the spring cultivars, accelerated development so that control and stressed plants flowered almost simultaneously. These genetic differences in development were correlated with a differential down-regulation of the flowering promotors FLOWERING LOCUS T1 and the BARLEY MADS-box genes BM3 and BM8. Our findings therefore demonstrate that Ppd-H1 affects developmental plasticity in response to drought in barley., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

FLOWERING LOCUS T-like (FT-like) genes control the photoperiodic regulation of flowering in many angiosperm plants. The family of FT-like genes is characterized by extensive gene duplication and subsequent diversification of FT functions which occurred independently in modern angiosperm lineages. In barley, there are 12 known FT-like genes (HvFT), but the function of most of them remains uncharacterized. This study aimed to characterize the role of HvFT4 in flowering time control and development in barley. The overexpression of HvFT4 in the spring cultivar Golden Promise delayed flowering time under long-day conditions. Microscopic dissection of the shoot apical meristem revealed that overexpression of HvFT4 specifically delayed spikelet initiation and reduced the number of spikelet primordia and grains per spike. Furthermore, ectopic overexpression of HvFT4 was associated with floret abortion and with the down-regulation of the barley MADS-box genes VRN-H1, HvBM3, and HvBM8 which promote floral development. This suggests that HvFT4 functions as a repressor of reproductive development in barley. Unraveling the genetic basis of FT-like genes can contribute to the identification of novel breeding targets to modify reproductive development and thereby spike morphology and grain yield., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Barley possesses a branchless, spike-shaped inflorescence where determinate spikelets attach directly to the main axis, but the developmental mechanism of spikelet identity remains largely unknown. Here we report the functional analysis of the barley gene BRANCHED AND INDETERMINATE SPIKELET 1 (BDI1), which encodes a TCP transcription factor and plays a crucial role in determining barley inflorescence architecture and spikelet development. The bdi1 mutant exhibited indeterminate spikelet meristems that continued to grow and differentiate after producing a floret meristem; some spikelet meristems at the base of the spike formed two fully developed seeds or converted to branched spikelets, producing a branched inflorescence. Map-based cloning analysis showed that this mutant has a deletion of ~600 kb on chromosome 5H containing three putative genes. Expression analysis and virus-induced gene silencing confirmed that the causative gene, BDI1, encodes a CYC/TB1-type TCP transcription factor and is highly conserved in both wild and cultivated barley. Transcriptome and regulatory network analysis demonstrated that BDI1 may integrate regulation of gene transcription cell wall modification and known trehalose-6-phosphate homeostasis to control spikelet development. Together, our findings reveal that BDI1 represents a key regulator of inflorescence architecture and meristem determinacy in cereal crop plants., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Potassium (K+) is the major cationic inorganic nutrient utilized for osmotic regulation, cell growth, and enzyme activation in plants. Inwardly rectifying K+ channel 1 (AKT1) is the primary channel for root K+ uptake in plants, but the function of HvAKT1 in barley plants under drought stress has not been fully elucidated. In this study, we conducted evolutionary bioinformatics, biotechnological, electrophysiological, and biochemical assays to explore molecular mechanisms of HvAKT1 in response to drought in barley. The expression of HvAKT1 was significantly up-regulated by drought stress in the roots of XZ5-a drought-tolerant wild barley genotype. We isolated and functionally characterized the plasma membrane-localized HvAKT1 using Agrobacterium-mediated plant transformation and Barley stripe mosaic virus-induced gene silencing of HvAKT1 in barley. Evolutionary bioinformatics indicated that the K+ selective filter in AKT1 originated from streptophyte algae and is evolutionarily conserved in land plants. Silencing of HvAKT1 resulted in significantly decreased biomass and suppressed K+ uptake in root epidermal cells under drought treatment. Disruption of HvAKT1 decreased root H+ efflux, H+-ATPase activity, and nitric oxide (NO) synthesis, but increased hydrogen peroxide (H2O2) production in the roots under drought stress. Furthermore, we observed that overexpression of HvAKT1 improves K+ uptake and increases drought resistance in barley. Our results highlight the importance of HvAKT1 for root K+ uptake and its pleiotropic effects on root H+-ATPase, and H2O2 and NO in response to drought stress, providing new insights into the genetic basis of drought tolerance and K+ nutrition in barley., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Understanding the control of fertility is critical for crop yield and breeding; this is particularly important for hybrid breeding to capitalize upon the resultant hybrid vigour. Different hybrid breeding systems have been adopted; however, these are challenging and crop specific. Mutants with environmentally reversible fertility offer valuable opportunities for hybrid breeding. The barley HvMS1 gene encodes a PHD-finger transcription factor that is expressed in the anther tapetum, which is essential for pollen development and causes complete male sterility when overexpressed in barley. This male sterility is due at least in part to indehiscent anthers resulting from incomplete tapetum degeneration, failure of anther opening, and sticky pollen under normal growth conditions (15 °C). However, dehiscence and fertility are restored when plants are grown at temperatures >20 °C, or when transferred to >20 °C during flowering prior to pollen mitosis I, with transfer at later stages unable to rescue fertility in vivo. As far as we are aware, this is the first report of thermosensitive male sterility in barley. This offers opportunities to understand the impact of temperature on pollen development and potential applications for environmentally switchable hybrid breeding systems; it also provides a 'female' male-sterile breeding tool that does not need emasculation to facilitate backcrossing., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Aluminum (Al) toxicity is a major abiotic stress that restricts crop production in acid soils. Plants have evolved internal and external mechanisms of tolerance, and among them it is well known that AtSTOP1 and OsART1 are key transcription factors involved in tolerance through regulation of multiple downstream genes. Here, we identified the closest homolog of these two proteins in barley, namely HvATF1, Al-tolerance Transcription Factor 1, and determined its potential function in Al stress. HvATF1 is expressed in the nucleus, and functions in transcriptional activation. The transcription of HvATF1 was found to be constitutive in different tissues, and was little affected by Al stress. Knockdown of HvATF1 by RNAi resulted in increased Al sensitivity. Transcriptomics analysis identified 64 differently expressed genes in the RNAi lines compared to the wild-type, and these were considered as candidate downstream genes regulated by HvATF1. This study provides insights into the different molecular mechanisms of Al tolerance in barley and other plants., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Aluminum (Al) toxicity is the primary limiting factor of crop production on acid soils. Tibetan wild barley germplasm is a valuable source of potential genes for breeding barley with acid and Al tolerance. We performed microRNA and RNA sequencing using wild (XZ16, Al-tolerant; XZ61, Al-sensitive) and cultivated (Dayton, Al-tolerant) barley. A novel homeobox-leucine zipper transcription factor, HvHOX9, was identified as a target gene of miR166b and functionally characterized. HvHOX9 was up-regulated by Al stress in XZ16 (but unchanged in XZ61 and Dayton) and was significantly induced only in root tip. Phylogenetic analysis showed that HvHOX9 is most closely related to wheat TaHOX9 and orthologues of HvHOX9 are present in the closest algal relatives of Zygnematophyceae. Barley stripe mosaic virus-induced gene silencing of HvHOX9 in XZ16 led to significantly increased Al sensitivity but did not affect its sensitivity to other metals and low pH. Disruption of HvHOX9 did not change Al concentration in the root cell sap, but led to more Al accumulation in root cell wall after Al exposure. Silencing of HvHOX9 decreased H+ influx after Al exposure. Our findings suggest that miR166b/HvHOX9 play a critical role in Al tolerance by decreasing root cell wall Al binding and increasing apoplastic pH for Al detoxification in the root., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The increasing demand for global food security in the face of a warming climate is leading researchers to investigate the physiological and molecular responses of cereals to rising ambient temperatures. Wheat and barley are temperate cereals whose yields are adversely affected by high ambient temperatures, with each 1 °C increase above optimum temperatures reducing productivity by 5-6%. Reproductive development is vulnerable to high-temperature stress, which reduces yields by decreasing grain number and/or size and weight. In recent years, analysis of early inflorescence development and genetic pathways that control the vegetative to floral transition have elucidated molecular processes that respond to rising temperatures, including those involved in the vernalization- and photoperiod-dependent control of flowering. In comparison, our understanding of genes that underpin thermal responses during later developmental stages remains poor, thus highlighting a key area for future research. This review outlines the responses of developmental genes to warmer conditions and summarizes our knowledge of the reproductive traits of wheat and barley influenced by high temperatures. We explore ways in which recent advances in wheat and barley research capabilities could help identify genes that underpin responses to rising temperatures, and how improved knowledge of the genetic regulation of reproduction and plant architecture could be used to develop thermally resilient cultivars., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Aphids secrete diverse repertoires of effectors into their hosts to promote the infestation process. While 'omics' approaches facilitated the identification and comparison of effector repertoires from a number of aphid species, the functional characterization of these proteins has been limited to dicot (model) plants. The bird cherry-oat aphid Rhopalosiphum padi is a pest of cereal crops, including barley. Here, we extend efforts to characterize aphid effectors with regard to their role in promoting susceptibility to the R. padi-barley interaction. We selected three R. padi effectors based on sequence similarity to previously characterized Myzus persicae effectors and assessed their subcellular localization, expression, and role in promoting plant susceptibility. Expression of R. padi effectors RpC002 and Rp1 in transgenic barley lines enhanced plant susceptibility to R. padi but not M. persicae, for which barley is a poor host. Characterization of Rp1 transgenic barley lines revealed reduced gene expression of plant hormone signalling genes relevant to plant-aphid interactions, indicating that this effector enhances susceptibility by suppressing plant defences in barley. Our data suggest that some aphid effectors specifically function when expressed in host species, and feature activities that benefit their corresponding aphid species., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Barley, like other crops, has experienced a series of genetic changes that have impacted its architecture and growth habit to suit the needs of humans, termed the domestication syndrome. Domestication also resulted in a concomitant bottleneck that reduced sequence diversity in genes and regulatory regions. Little is known about regulatory changes resulting from domestication in barley. We used RNA sequencing to examine allele-specific expression in hybrids between wild and domesticated barley. Our results show that most genes have conserved regulation. In contrast to studies of allele-specific expression in interspecific hybrids, we find almost a complete absence of trans effects. We also find that cis regulation is largely stable in response to short-term cold stress. Our study has practical implications for crop improvement using wild relatives. Genes regulated in cis are more likely to be expressed in a new genetic background at the same level as in their native background., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Mobilization of reserves in germinated cereal grains is critical for early seedling vigour, global crop productivity, and hence food security. Gibberellins (GAs) are central to this process. We have developed a spatio-temporal model that describes the multifaceted mechanisms of GA regulation in germinated barley grain. The model was generated using RNA sequencing transcript data from tissues dissected from intact, germinated grain, which closely match measurements of GA hormones and their metabolites in those tissues. The data show that successful grain germination is underpinned by high concentrations of GA precursors in ungerminated grain, the use of independent metabolic pathways for the synthesis of several bioactive GAs during germination, and a capacity to abort bioactive GA biosynthesis. The most abundant bioactive form is GA1, which is synthesized in the scutellum as a glycosyl conjugate that diffuses to the aleurone, where it stimulates de novo synthesis of a GA3 conjugate and GA4. Synthesis of bioactive GAs in the aleurone provides a mechanism that ensures the hormonal signal is relayed from the scutellum to the distal tip of the grain. The transcript data set of 33 421 genes used to define GA metabolism is available as a resource to analyse other physiological processes in germinated grain., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Flowering time is a complex trait and has a key role in crop yield and adaptation to environmental stressors such as heat and drought. This study aimed to better understand the interconnected dynamics of epistasis and environment and look for novel regulators. We investigated 534 spring barley MAGIC DH lines for flowering time at various environments. Analysis of quantitative trait loci (QTLs), epistatic interactions, QTL × environment (Q×E) interactions, and epistasis × environment (E×E) interactions were performed with single SNP and haplotype approaches. In total, 18 QTLs and 2420 epistatic interactions were detected, including intervals harboring major genes such as Ppd-H1, Vrn-H1, Vrn-H3, and denso/sdw1. Epistatic interactions found in field and semi-controlled conditions were distinctive. Q×E and E×E interactions revealed that temperature influenced flowering time by triggering different interactions between known and newly detected regulators. A novel flowering-delaying QTL allele was identified on chromosome 1H (named 'HvHeading') and was shown to be engaged in epistatic and environment interactions. Results suggest that investigating epistasis, environment, and their interactions, rather than only single QTLs, is an effective approach for detecting novel regulators. We assume that barley can adapt flowering time to the environment via alternative routes within the pathway., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

In cereal grain, sucrose is converted into storage carbohydrates: mainly starch, fructan, and mixed-linkage (1,3;1,4)-β-glucan (MLG). Previously, endosperm-specific overexpression of the HvCslF6 gene in hull-less barley was shown to result in high MLG and low starch content in mature grains. Morphological changes included inwardly elongated aleurone cells, irregular cell shapes of peripheral endosperm, and smaller starch granules of starchy endosperm. Here we explored the physiological basis for these defects by investigating how changes in carbohydrate composition of developing grain impact mature grain morphology. Augmented MLG coincided with increased levels of soluble carbohydrates in the cavity and endosperm at the storage phase. Transcript levels of genes relating to cell wall, starch, sucrose, and fructan metabolism were perturbed in all tissues. The cell walls of endosperm transfer cells (ETCs) in transgenic grain were thinner and showed reduced mannan labelling relative to the wild type. At the early storage phase, ruptures of the non-uniformly developed ETCs and disorganization of adjacent endosperm cells were observed. Soluble sugars accumulated in the developing grain cavity, suggesting a disturbance of carbohydrate flow from the cavity towards the endosperm, resulting in a shrunken mature grain phenotype. Our findings demonstrate the importance of regulating carbohydrate partitioning in maintenance of grain cellularization and filling processes., (© Society for Experimental Biology 2019.)

The biosynthesis of starch granules in plant plastids is coordinated by the orchestrated action of transferases, hydrolases, and dikinases. These enzymes either contain starch-binding domain(s) themselves, or are dependent on direct interactions with co-factors containing starch-binding domains. As a means to competitively interfere with existing starch-protein interactions, we expressed the protein module Carbohydrate-Binding Motif 20 (CBM20), which has a very high affinity for starch, ectopically in barley plastids. This interference resulted in an increase in the number of starch granules in chloroplasts and in formation of compound starch granules in grain amyloplasts, which is unusual for barley. More importantly, we observed a photosystem-independent inhibition of CO2 fixation, with a subsequent reduced growth rate and lower accumulation of carbohydrates with effects throughout the metabolome, including lower accumulation of transient leaf starch. Our results demonstrate the importance of endogenous starch-protein interactions for controlling starch granule morphology and number, and plant growth, as substantiated by a metabolic link between starch-protein interactions and control of CO2 fixation in chloroplasts., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology.)