Over-accumulation of chloroplast-nucleus located WHIRLY1 in barley leads to a decrease in growth and an enhanced stress resistance.

WHIRLY1 is a chloroplast-nucleus located DNA/RNA-binding protein with functions in development and stress tolerance. By overexpression of HvWHIRLY1 in barley, one line with a 10-fold and two lines with a 50-fold accumulation of the protein were obtained. In these lines, the relative abundance of the nuclear form exceeded that of the chloroplast form. Growth of the plants was shown to be compromised in a WHIRLY1 abundance-dependent manner. Over-accumulation of WHIRLY1 in chloroplasts had neither an evident impact on nucleoid morphology nor on the composition of the photosynthetic apparatus. Nevertheless, oeW1 plants were found to be compromised in the light reactions of photosynthesis as well as in carbon fixation. The reduction in growth and photosynthesis was shown to be accompanied by a decrease in the levels of cytokinins and an increase in the level of jasmonic acid. Gene expression analyses revealed that in nonstress conditions the oeW1 plants had enhanced levels of pathogen response (PR) gene expression indicating activation of constitutive defense. During growth in continuous light of high irradiance PR gene expression increased indicating that under stress conditions oeW1 are capable to further enhance defense.

A novel type of malformed floral organs mutant in barley was conferred by loss-of-function mutations of the MADS-box gene HvAGL6.

The advanced model of floral morphogenesis is based largely on data from Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), but this process is less well understood in the Triticeae. Here, we investigated a sterile barley (Hordeum vulgare) mutant with malformed floral organs (designated mfo1), of which the paleae, lodicules, and stamens in each floret were all converted into lemma-like organs, and the ovary was abnormally shaped. Combining bulked-segregant analysis, whole-genome resequencing, and TILLING approaches, the mfo1 mutant was attributed to loss-of-function mutations in the MADS-box transcription factor gene HvAGL6, a key regulator in the ABCDE floral morphogenesis model. Through transcriptomic analysis between young inflorescences of wild-type and mfo1 plants, 380 genes were identified as differentially expressed, most of which function in DNA binding, protein dimerization, cell differentiation, or meristem determinacy. Regulatory pathway enrichment showed HvAGL6 associates with transcriptional abundance of many MADS-box genes, including the B-class gene HvMADS4. Mutants with deficiency in HvMADS4 exhibited the conversion of stamens into supernumerary pistils, producing multiple ovaries resembling the completely sterile multiple ovaries 3.h (mov3.h) mutant. These findings demonstrate that the regulatory model of floral morphogenesis is conserved across plant species and provides insights into the interactions between HvAGL6 and other MADS-box regulators.

Hordeum vulgare CYP76M57 catalyzes C2 shortening of tryptophan side chain by C-N bond rearrangement in gramine biosynthesis.

The indole alkaloid gramine, 3-(dimethylaminomethyl)indole, is a defensive specialized metabolite found in some barley cultivars. In its biosynthetic process, the tryptophan (Trp) side chain is shortened by two carbon atoms to produce 3-(aminomethyl)indole (AMI), which is then methylated by N-methyltransferase (HvNMT) to produce gramine. Although side chain shortening is one of the crucial scaffold formation steps of alkaloids originating from aromatic amino acids, the gene and enzyme involved in the Trp-AMI conversion reactions are unknown. In this study, through RNA-seq analysis, 35 transcripts were shown to correlate with gramine production; among them, an uncharacterized cytochrome P450 (CYP) gene, CYP76M57, and HvNMT were identified as candidate genes for gramine production. Transgenic Arabidopsis thaliana and rice overexpressing CYP and HvNMT accumulate AMI, N-methyl-AMI, and gramine. CYP76M57, heterologously expressed in Pichia pastoris, was able to act on Trp to produce AMI. Furthermore, the amino group nitrogen of Trp was retained during the CYP76M57-catalyzed reaction, indicating that the C2 shortening of Trp proceeds with an unprecedented biosynthetic process, the removal of the carboxyl group and Cα and the rearrangement of the nitrogen atom to Cß. In some gramine-non-accumulating barley cultivars, arginine 104 in CYP76M57 is replaced by threonine, which abolished the catalytic activity of CYP76M57 to convert Trp into AMI. These results uncovered the missing committed enzyme of gramine biosynthesis in barley and contribute to the elucidation of the potential functions of CYPs in plants and undiscovered specialized pathways.

HvGST plays a key role in anthocyanin accumulation in colored barley.

Blue aleurone of barley is caused by the accumulation of delphinidin-based derivatives. Although these compounds are ideal nutrients for human health, they are undesirable contaminants in malt brewing. Therefore, the ability to add and remove this trait easily would facilitate breeding barley for different purposes. Here we identified a glutathione S-transferase gene (HvGST) that was responsible for the blue aleurone trait in Tibetan qingke barley by performing a genome-wide association study and RNA-sequencing analysis. Gene variation and expression analysis indicated that HvGST also participates in the transport and accumulation of anthocyanin in purple barley. Haplotype and the geographic distribution analyses of HvGST alleles revealed two independent natural variants responsible for the emergence of white aleurone: a 203-bp deletion causing premature termination of translation in qingke barley and two key single nucleotide polymorphisms in the promoter resulting in low transcription in Western barley. This study contributes to a better understanding of mechanisms of colored barley formation, and provides a comprehensive reference for marker-assisted barley breeding.

Molecular identification of a laccase that catalyzes the oxidative coupling of a hydroxycinnamic acid amide for hordatine biosynthesis in barley.

Plants produce dimerized phenolic compounds as secondary metabolites. Hordatine A (HA), a dehydrodimer of p-coumaroylagmatine (pCA), is an antifungal compound accumulated at high levels in young barley (Hordeum vulgare) seedlings. The enzyme responsible for the oxidative dimerization of pCA, which is the final step of the hordatine biosynthetic pathway, has not been identified. In this study, we first verified the presence of this enzyme activity in the crude extract of barley seedlings. Because the enzyme activity was not dependent on H2 O2 , the responsible enzyme was not peroxidase, which was previously implicated in HA biosynthesis. The analysis of the dissection lines of wheat (Triticum aestivum) carrying aberrant barley 2H chromosomes detected HA in the wheat lines carrying the distal part of the 2H short arm. This chromosomal region contains two laccase genes (HvLAC1 and HvLAC2) that are highly expressed at the seedling stage and may encode enzymes that oxidize pCA during the formation of HA. Changes in the HvLAC transcript levels coincided with the changes in the HA biosynthesis-related enzyme activities in the crude extract and the HA content in barley seedlings. Moreover, HvLAC genes were heterologously expressed in Nicotiana benthamiana leaves and in bamboo (Phyllostachys nigra) suspension cells and HA biosynthetic activities were detected in the crude extract of transformed N. benthamiana leaves and bamboo suspension cells. The HA formed by the enzymatic reaction had the same stereo-configuration as the naturally occurring HA. These results demonstrate that HvLAC enzymes mediate the oxidative coupling of pCA during HA biosynthesis.

Spatial organization and dynamics of chromosomes and microtubules during barley mitosis.

Mitosis and cytokinesis are fundamental processes through which somatic cells increase their numbers and allow plant growth and development. Here, we analyzed the organization and dynamics of mitotic chromosomes, nucleoli, and microtubules in living cells of barley root primary meristems using a series of newly developed stable fluorescent protein translational fusion lines and time-lapse confocal microscopy. The median duration of mitosis from prophase until the end of telophase was 65.2 and 78.2 min until the end of cytokinesis. We showed that barley chromosomes frequently start condensation before mitotic pre-prophase as defined by the organization of microtubules and maintain it even after entering into the new interphase. Furthermore, we found that the process of chromosome condensation does not finish at metaphase, but gradually continues until the end of mitosis. In summary, our study features resources for in vivo analysis of barley nuclei and chromosomes and their dynamics during mitotic cell cycle.

Protein reservoirs of seeds are amyloid composites employed differentially for germination and seedling emergence.

Seed protein localization in seed storage protein bodies (SSPB) and their significance in germination are well recognized. SSPB are spherical and contain an assembly of water-soluble and salt-soluble proteins. Although the native structures of some SSPB proteins are explored, their structural arrangement to the functional correlation in SSPB remains unknown. SSPB are morphologically analogous to electron-dense amyloid-containing structures reported in other organisms. Here, we show that wheat, mungbean, barley, and chickpea SSPB exhibit a speckled pattern of amyloids interspersed in an amyloid-like matrix along with native structures, suggesting the composite nature of SSPB. This is confirmed by multispectral imaging methods, electron microscopy, infrared, and X-ray diffraction analysis, using in situ tissue sections, ex vivo protoplasts, and in vitro SSPB. Laser capture microdissection coupled with peptide fingerprinting has shown that globulin 1 and 3 in wheat, and 8S globulin and conglycinin in mungbean are the major amyloidogenic proteins. The amyloid composites undergo a sustained degradation during germination and seedling growth, facilitated by an intricate interplay of plant hormones and proteases. These results would lay down the foundation for understanding the amyloid composite structure during SSPB biogenesis and its evolution across the plant kingdom and have implications in both basic and applied plant biology.

Helical chromonema coiling is conserved in eukaryotes.

Efficient chromatin condensation is required to transport chromosomes during mitosis and meiosis, forming daughter cells. While it is well accepted that these processes follow fundamental rules, there has been a controversial debate for more than 140 years on whether the higher-order chromatin organization in chromosomes is evolutionarily conserved. Here, we summarize historical and recent investigations based on classical and modern methods. In particular, classical light microscopy observations based on living, fixed, and treated chromosomes covering a wide range of plant and animal species, and even in single-cell eukaryotes suggest that the chromatids of large chromosomes are formed by a coiled chromatin thread, named the chromonema. More recently, these findings were confirmed by electron and super-resolution microscopy, oligo-FISH, molecular interaction data, and polymer simulation. Altogether, we describe common and divergent features of coiled chromonemata in different species. We hypothesize that chromonema coiling in large chromosomes is a fundamental feature established early during the evolution of eukaryotes to handle increasing genome sizes.

Genome-wide identification and expression analysis of the SPL gene family and its response to abiotic stress in barley (Hordeum vulgare L.).

BACKGROUND: Squamosa promoter-binding protein-like (SPL) is a plant-specific transcription factor that is widely involved in the regulation of plant growth and development, including flower and grain development, stress responses, and secondary metabolite synthesis. However, this gene family has not been comprehensively evaluated in barley, the most adaptable cereal crop with a high nutritional value. RESULTS: In this study, a total of 15 HvSPL genes were identified based on the Hordeum vulgare genome. These genes were named HvSPL1 to HvSPL15 based on the chromosomal distribution of the HvSPL genes and were divided into seven groups (I, II, III, V, VI, VII, and VIII) based on the phylogenetic tree analysis. Chromosomal localization revealed one pair of tandem duplicated genes and one pair of segmental duplicated genes. The HvSPL genes exhibited the highest collinearity with the monocotyledonous plant, Zea mays (27 pairs), followed by Oryza sativa (18 pairs), Sorghum bicolor (16 pairs), and Arabidopsis thaliana (3 pairs), and the fewest homologous genes with Solanum lycopersicum (1 pair). The distribution of the HvSPL genes in the evolutionary tree was relatively scattered, and HvSPL proteins tended to cluster with SPL proteins from Z. mays and O. sativa, indicating a close relationship between HvSPL and SPL proteins from monocotyledonous plants. Finally, the spatial and temporal expression patterns of the 14 HvSPL genes from different subfamilies were determined using quantitative real-time polymerase chain reaction (qRT-PCR). Based on the results, the HvSPL gene family exhibited tissue-specific expression and played a regulatory role in grain development and abiotic stress. HvSPL genes are highly expressed in various tissues during seed development. The expression levels of HvSPL genes under the six abiotic stress conditions indicated that many genes responded to stress, especially HvSPL8, which exhibited high expression under multiple stress conditions, thereby warranting further attention. CONCLUSION: In this study, 15 SPL gene family members were identified in the genome of Hordeum vulgare, and the phylogenetic relationships, gene structure, replication events, gene expression, and potential roles of these genes in millet development were studied. Our findings lay the foundation for exploring the HvSPL genes and performing molecular breeding of barley.

Highly divergent satellitomes of two barley species of agronomic importance, Hordeum chilense and H. vulgare.

In this paper, we have performed an in-depth study of the complete set of the satellite DNA (satDNA) families (i.e. the satellitomes) in the genome of two barley species of agronomic value in a breeding framework, H. chilense (H1 and H7 accessions) and H. vulgare (H106 accession), which can be useful tools for studying chromosome associations during meiosis. The study has led to the analysis of a total of 18 satDNA families in H. vulgare, 25 satDNA families in H. chilense (accession H1) and 27 satDNA families in H. chilense (accession H7) that constitute 46 different satDNA families forming 36 homology groups. Our study highlights different important contributions of evolutionary and applied interests. Thus, both barley species show very divergent satDNA profiles, which could be partly explained by the differential effects of domestication versus wildlife. Divergence derives from the differential amplification of different common ancestral satellites and the emergence of new satellites in H. chilense, usually from pre-existing ones but also random sequences. There are also differences between the two H. chilense accessions, which support genetically distinct groups. The fluorescence in situ hybridization (FISH) patterns of some satDNAs yield distinctive genetic markers for the identification of specific H. chilense or H. vulgare chromosomes. Some of the satellites have peculiar structures or are related to transposable elements which provide information about their origin and expansion. Among these, we discuss the existence of different (peri)centromeric satellites that supply this region with some plasticity important for centromere evolution. These peri(centromeric) satDNAs and the set of subtelomeric satDNAs (a total of 38 different families) are analyzed in the framework of breeding as the high diversity found in the subtelomeric regions might support their putative implication in chromosome recognition and pairing during meiosis, a key point in the production of addition/substitution lines and hybrids.

Super-resolution microscopy reveals the number and distribution of topoisomerase IIα and CENH3 molecules within barley metaphase chromosomes.

Topoisomerase IIα (Topo IIα) and the centromere-specific histone H3 variant CENH3 are key proteins involved in chromatin condensation and centromere determination, respectively. Consequently, they are required for proper chromosome segregation during cell divisions. We combined two super-resolution techniques, structured illumination microscopy (SIM) to co-localize Topo IIα and CENH3, and photoactivated localization microscopy (PALM) to determine their molecule numbers in barley metaphase chromosomes. We detected a dispersed Topo IIα distribution along chromosome arms but an accumulation at centromeres, telomeres, and nucleolus-organizing regions. With a precision of 10-50 nm, we counted ~ 20,000-40,000 Topo IIα molecules per chromosome, 28% of them within the (peri)centromere. With similar precision, we identified ~13,500 CENH3 molecules per centromere where Topo IIα proteins and CENH3-containing chromatin intermingle. In short, we demonstrate PALM as a useful method to count and localize single molecules with high precision within chromosomes. The ultrastructural distribution and the detected amount of Topo IIα and CENH3 are instrumental for a better understanding of their functions during chromatin condensation and centromere determination.

Investigation of the morphological, physiological, biochemical, and catabolic characteristics and gene expression under drought stress in tolerant and sensitive genotypes of wild barley [Hordeum vulgare subsp. spontaneum (K. Koch) Asch. & Graebn.].

BACKGROUND: Barley (H. vulgare L.) is an important cereal crop cultivated across various climates globally. Barley and its ancestor (H. vulgare subsp. spontaneum) are an economically valuable model for genetic research and improvement. Drought, among various abiotic stresses, is a substantial threat to agriculture due to its unpredictable nature and significant impact on crop yield. RESULTS: This study was conducted in both greenhouse and laboratory settings. Prior to the study, wild barley accessions were pre-selected based on their sensitivity or tolerance to drought as determined from fieldwork in the 2020-2021 and 2021-2022 cropping seasons. The effects of three levels of drought stress were evaluated (control, 90-95% field capacity [FC]; mild stress, 50-55% FC; and severe stress, 25-30% FC). Several parameters were assessed, including seedling and root growth, enzymatic activity (CAT, SOD, POD), soluble protein levels, chlorophyll content, carotenoids, abaxial and adaxial stomatal density and dimensions, and relative gene expression of Dhn1, SOD, POD, and CAT. Drought stress significantly increased enzyme activities, especially at 25-30% FC, and more in the tolerant genotype. On the other hand, sensitive genotypes showed a notable increase in stomatal density. Under drought stress, there was a general decline in seedling and root growth, protein content, chlorophyll and carotenoids, and stomatal dimensions. Importantly, gene expression analysis revealed that Dhn1, SOD, POD, and CAT were upregulated under drought, with the highest expression levels observed in the drought-tolerant genotype under severe stress conditions (25-30% FC). CONCLUSIONS: Our investigation highlights the distinct morphological, physiological, biochemical, and gene-expression profiles of drought-resistant and drought-sensitive wild barley genotypes under varying degrees of drought.

Transcriptional changes during crown-root development and emergence in barley (Hordeum vulgare L.).

BACKGROUND: Roots play an important role during plant growth and development, ensuring water and nutrient uptake. Understanding the mechanisms regulating their initiation and development opens doors towards root system architecture engineering. RESULTS: Here, we investigated by RNA-seq analysis the changes in gene expression in the barley stem base of 1 day-after-germination (DAG) and 10DAG seedlings when crown roots are formed. We identified 2,333 genes whose expression was lower in the stem base of 10DAG seedlings compared to 1DAG seedlings. Those genes were mostly related to basal cellular activity such as cell cycle organization, protein biosynthesis, chromatin organization, cytoskeleton organization or nucleotide metabolism. In opposite, 2,932 genes showed up-regulation in the stem base of 10DAG seedlings compared to 1DAG seedlings, and their function was related to phytohormone action, solute transport, redox homeostasis, protein modification, secondary metabolism. Our results highlighted genes that are likely involved in the different steps of crown root formation from initiation to primordia differentiation and emergence, and revealed the activation of different hormonal pathways during this process. CONCLUSIONS: This whole transcriptomic study is the first study aiming at understanding the molecular mechanisms controlling crown root development in barley. The results shed light on crown root emergence that is likely associated with a strong cell wall modification, death of the cells covering the crown root primordium, and the production of defense molecules that might prevent pathogen infection at the site of root emergence.

Transcriptome and metabolome analyses reveal molecular insights into waterlogging tolerance in Barley.

Waterlogging stress is one of the major abiotic stresses affecting the productivity and quality of many crops worldwide. However, the mechanisms of waterlogging tolerance are still elusive in barley. In this study, we identify key differentially expressed genes (DEGs) and differential metabolites (DM) that mediate distinct waterlogging tolerance strategies in leaf and root of two barley varieties with contrasting waterlogging tolerance under different waterlogging treatments. Transcriptome profiling revealed that the response of roots was more distinct than that of leaves in both varieties, in which the number of downregulated genes in roots was 7.41-fold higher than that in leaves of waterlogging sensitive variety after 72 h of waterlogging stress. We also found the number of waterlogging stress-induced upregulated DEGs in the waterlogging tolerant variety was higher than that of the waterlogging sensitive variety in both leaves and roots in 1 h and 72 h treatment. This suggested the waterlogging tolerant variety may respond more quickly to waterlogging stress. Meanwhile, phenylpropanoid biosynthesis pathway was identified to play critical roles in waterlogging tolerant variety by improving cell wall biogenesis and peroxidase activity through DEGs such as Peroxidase (PERs) and Cinnamoyl-CoA reductases (CCRs) to improve resistance to waterlogging. Based on metabolomic and transcriptomic analysis, we found the waterlogging tolerant variety can better alleviate the energy deficiency via higher sugar content, reduced lactate accumulation, and improved ethanol fermentation activity compared to the waterlogging sensitive variety. In summary, our results provide waterlogging tolerance strategies in barley to guide the development of elite genetic resources towards waterlogging-tolerant crop varieties.

Dissecting the resilience of barley genotypes under multiple adverse environmental conditions.

As climate change increases abiotic stresses like drought and heat, evaluating barley performance under such conditions is critical for maintaining productivity. To assess how barley performs under normal conditions, drought, and heat stress, 29 different varieties were examined, considering agronomic, physiological, and disease-related characteristics. The research was conducted in five environments: two normal environments in 2020/2021 and 2021/2022, two drought stress environments in 2020/2021 and 2021/2022, and one heat stress environment in 2021/2022. The results demonstrated that genotype and environment significantly influenced all traits (p < 0.05), except canopy temperature, while genotype x environment interaction significantly influenced most traits, except total chlorophyll content and canopy temperature. Heat and drought stress environments often resulted in reduced performance for traits like plant height, spike length, grains per spike, and 100-grain weight compared to normal conditions. Based on individual traits, genotypes 07UT-44, 06WA-77, 08AB-09, and 07N6-57 exhibited the highest grain yield (4.1, 3.6, 3.6, and 3.6 t/ha, respectively). Also, these genotypes demonstrated enhanced stability in diverse drought and heat stress conditions, as assessed by the mean performance vs. stability index (Weighted Average of Absolute Scores, WAASB). The multi-trait stability index (MTSI) identified 07UT-44, 07UT-55, 07UT-71, and 08AB-09 as the most stable genotypes in terms of the performance of all traits. The imported lines demonstrated superior performance and stability, highlighting their potential as valuable genetic resources for developing climate-resilient barley.

Silicon regulates phosphate deficiency through involvement of auxin and nitric oxide in barley roots.

MAIN CONCLUSION: Silicon application mitigates phosphate deficiency in barley through an interplay with auxin and nitric oxide, enhancing growth, photosynthesis, and redox balance, highlighting the potential of silicon as a fertilizer for overcoming nutritional stresses. Silicon (Si) is reported to attenuate nutritional stresses in plants, but studies on the effect of Si application to plants grown under phosphate (Pi) deficiency are still very scarce, especially in barley. Therefore, the present work was undertaken to investigate the potential role of Si in mitigating the adverse impacts of Pi deficiency in barley Hordeum vulgare L. (var. BH902). Further, the involvement of two key regulatory signaling molecules--auxin and nitric oxide (NO)--in Si-induced tolerance against Pi deficiency in barley was tested. Morphological attributes, photosynthetic parameters, oxidative stress markers (O2·-, H2O2, and MDA), antioxidant system (enzymatic--APX, CAT, SOD, GR, DHAR, MDHAR as well as non-enzymatic--AsA and GSH), NO content, and proline metabolism were the key traits that were assessed under different treatments. The P deficiency distinctly declined growth of barley seedlings, which was due to enhancement in oxidative stress leading to inhibition of photosynthesis. These results were also in parallel with an enhancement in antioxidant activity, particularly SOD and CAT, and endogenous proline level and its biosynthetic enzyme (P5CS). The addition of Si exhibited beneficial effects on barley plants grown in Pi-deficient medium as reflected in increased growth, photosynthetic activity, and redox balance through the regulation of antioxidant machinery particularly ascorbate-glutathione cycle. We noticed that auxin and NO were also found to be independently participating in Si-mediated improvement of growth and other parameters in barley roots under Pi deficiency. Data of gene expression analysis for PHOSPHATE TRANSPORTER1 (HvPHT1) indicate that Si helps in increasing Pi uptake as per the need of Pi-deficient barley seedlings, and also auxin and NO both appear to help Si in accomplishing this task probably by inducing lateral root formation. These results are suggestive of possible application of Si as a fertilizer to correct the negative effects of nutritional stresses in plants. Further research at genetic level to understand Si-induced mechanisms for mitigating Pi deficiency can be helpful in the development of new varieties with improved tolerance against Pi deficiency, especially for cultivation in areas with Pi-deficient soils.

Disentangling sources of gene tree discordance for Hordeum species via target-enriched sequencing assays.

Hordeum is an economically and evolutionarily important genus within the Triticeae tribe of the family Poaceae, and contains 33 widely distributed and diverse species which cytologically represent four subgenomes (H, Xa, Xu and I). These wild species (except Hordeum spontaneum, which is the primary gene pool of barley) are secondary or tertiary gene-pool germplasms for barley and wheat improvement, and uncovering their complicated evolutionary relationships would benefit for future breeding programs. Here, we developed a complexity-reduced pipeline via capturing genome-wide distributed fragments via two novel target-enriched assays (HorCap v1.0 and BarPlex v1.0) in conjugation with high-throughput sequencing of the enrichments. Both assays were tested for genotyping 40 species from three genera (Hordeum, Triticum, and Aegilops) containing 82 samples 67 accessions. Either of both assays worked efficiently in genotyping, while integration of both assays can significantly improve the robustness and resolution of the Hordeum phylogenetic trees. Interestingly, the incomplete lineage sorting (ILS) was inferred for the first time as the major factor causing phylogenetic discordance among the four subgenomes, whereas in New World species (carrying I genome) post-speciation introgression events were revealed. Through revising the evolutionary relationships of the Hordeum species based on an ancestral state reconstruction for the diploids and parental donor inference for the polyploids, our results raised new queries about the Hordeum phylogeny. Moreover, both newly-developed assays are applicable in genotyping and phylogenetic analysis of Hordeum and other Triticeae wild species.

Jasmonates, gibberellins, and powdery mildew modify cell cycle progression and evoke differential spatiotemporal responses along the barley leaf.

Barley (Hordeum vulgare) is an important cereal crop, and its development, defence, and stress responses are modulated by different hormones including jasmonates (JAs) and the antagonistic gibberellins (GAs). Barley productivity is severely affected by the foliar biotrophic fungal pathogen Blumeria hordei. In this study, primary leaves were used to examine the molecular processes regulating responses to methyl-jasmonate (MeJA) and GA to B. hordei infection along the leaf axis. Flow cytometry, microscopy, and spatiotemporal expression patterns of genes associated with JA, GA, defence, and the cell cycle provided insights on cell cycle progression and on the gradient of susceptibility to B. hordei observed along the leaf. Notably, the combination of B. hordei with MeJA or GA pre-treatment had a different effect on the expression patterns of the analysed genes compared to individual treatments. MeJA reduced susceptibility to B. hordei in the proximal part of the leaf blade. Overall, distinctive spatiotemporal gene expression patterns correlated with different degrees of cell proliferation, growth capacity, responses to hormones, and B. hordei infection along the leaf. Our results highlight the need to further investigate differential spatial and temporal responses to pathogens at the organ, tissue, and cell levels in order to devise effective disease control strategies in crops.

Contrasting cytosolic glutathione redox dynamics under abiotic and biotic stress in barley as revealed by the biosensor Grx1-roGFP2.

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.

HOMEOBOX2, the paralog of SIX-ROWED SPIKE1/HOMEOBOX1, is dispensable for barley spikelet development.

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.