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Global transcriptome studies demonstrated the existence of unique plant responses under combined stress which are otherwise not seen during individual stresses. In order to combat combined stress plants use signaling pathways and ‘cross talk’ mediated by hormones involved in stress and growth related processes. However, interactions among hormones’ pathways in combined stressed plants are not yet known. Here we studied dynamics of different hormones under individual and combined drought and pathogen infection in Arabidopsis thaliana by liquid chromatography-mass spectrometry (LC-MS) based profiling. Our results revealed abscisic acid (ABA) and salicylic acid (SA) as key regulators under individual drought and pathogen stress respectively. Under combined drought and host pathogen stress (DH) we observed non-induced levels of ABA with an upsurge in SA and jasmonic acid (JA) concentrations, underscoring their role in basal tolerance against host pathogen. Under a non-host pathogen interaction with drought (DNH) stressed plants, ABA, SA and JA profiles were similar to those under DH or non-host pathogen alone. We propose that plants use SA/JA dependent signaling during DH stress which antagonize ABA biosynthesis and signaling pathways during early stage of stress. The study provides insights into hormone modulation at different time points during combined stress.

Different plant cultivars of the same genus and species can exhibit vastly different genetic transformation efficiencies. However, the genetic factors underlying these differences in transformation rate remain largely unknown. In barley, ‘Golden Promise’ is one of a few cultivars reliable for Agrobacterium -mediated transformation. By contrast, cultivar ‘Haruna Nijo’ is recalcitrant to genetic transformation. We identified genomic regions of barley important for successful transformation with Agrobacterium , utilizing the ‘Haruna Nijo’ × ‘Golden Promise’ F 2 generation and genotyping by 124 genome-wide SNP markers. We observed significant segregation distortions of these markers from the expected 1:2:1 ratio toward the ‘Golden Promise’-type in regions of chromosomes 2H and 3H, indicating that the alleles of ‘Golden Promise’ in these regions might contribute to transformation efficiency. The same regions, which we termed Transformation Amenability (TFA ) regions, were also conserved in transgenic F 2 plants generated from a ‘Morex’ × ‘Golden Promise’ cross. The genomic regions identified herein likely include necessary factors for Agrobacterium -mediated transformation in barley. The potential to introduce these loci into any haplotype of barley opens the door to increasing the efficiency of transformation for target alleles into any haplotype of barley by the TFA -based methods proposed in this report.

Summary Ferulate 5‐hydroxylase (F5H) catalyses the hydroxylation of coniferyl alcohol and coniferaldehyde for the biosynthesis of syringyl (S) lignin in angiosperms. However, the coordinated effects of F5H with caffeic acid O‐methyltransferase (COMT) on the metabolic flux towards S units are largely unknown. We concomitantly regulated F5H expression in COMT‐down‐regulated transgenic switchgrass (Panicum virgatum L.) lines and studied the coordination of F5H and COMT in lignin biosynthesis. Down‐regulation of F5H in COMT‐RNAi transgenic switchgrass plants further impeded S lignin biosynthesis and, consequently, increased guaiacyl (G) units and reduced 5‐OH G units. Conversely, overexpression of F5H in COMT‐RNAi transgenic plants reduced G units and increased 5‐OH units, whereas the deficiency of S lignin biosynthesis was partially compensated or fully restored, depending on the extent of COMT down‐regulation in switchgrass. Moreover, simultaneous regulation of F5H and COMT expression had different effects on cell wall digestibility of switchgrass without biomass loss. Our results indicate that up‐regulation and down‐regulation of F5H expression, respectively, have antagonistic and synergistic effects on the reduction in S lignin resulting from COMT suppression. The coordinated effects between lignin genes should be taken into account in future studies aimed at cell wall bioengineering.

Background Sequencing analysis of mitochondrial genomes is important for understanding the evolution and genome structures of various plant species. Barley is a self-pollinated diploid plant with seven chromosomes comprising a large haploid genome of 5.1 Gbp. Wild barley ( Hordeum vulgare ssp. spontaneum ) and cultivated barley ( H. vulgare ssp. vulgare ) have cross compatibility and closely related genomes, although a significant number of nucleotide polymorphisms have been reported between their genomes. Results We determined the complete nucleotide sequences of the mitochondrial genomes of wild and cultivated barley. Two independent circular maps of the 525,599 bp barley mitochondrial genome were constructed by de novo assembly of high-throughput sequencing reads of barley lines H602 and Haruna Nijo, with only three SNPs detected between haplotypes. These mitochondrial genomes contained 33 protein-coding genes, three ribosomal RNAs, 16 transfer RNAs, 188 new ORFs, six major repeat sequences and several types of transposable elements. Of the barley mitochondrial genome-encoded proteins, NAD6, NAD9 and RPS4 had unique structures among grass species. Conclusions The mitochondrial genome of barley was similar to those of other grass species in terms of gene content, but the configuration of the genes was highly differentiated from that of other grass species. Mitochondrial genome sequencing is essential for annotating the barley nuclear genome; our mitochondrial sequencing identified a significant number of fragmented mitochondrial sequences in the reported nuclear genome sequences. Little polymorphism was detected in the barley mitochondrial genome sequences, which should be explored further to elucidate the evolution of barley.

Background QTL-seq, in combination with bulked segregant analysis and next-generation sequencing (NGS), is used to identify loci in small plant genomes, but is technically challenging to perform in species with large genomes, such as barley. A combination of exome sequencing and QTL-seq (exome QTL-seq) was used to map the mono-factorial Mendelian locus black lemma and pericarp ( Blp ) and QTLs for resistance to net blotch disease, a common disease of barley caused by the fungus Pyrenophora teres , which segregated in a population of 100 doubled haploid barley lines. Methods The provisional exome sequences were prepared by ordering the loci of expressed genes based on the genome information and concatenating genes with intervals of 200-bp spacer \"N\" for each chromosome. The QTL-seq pipeline was used to analyze short reads from the exome-captured library. Results In this study, short NGS reads of bulked total DNA samples from segregants with extreme trait values were subjected to exome capture, and the resulting exome sequences were aligned to the reference genome. SNP allele frequencies were compared to identify the locations of genes/QTLs responsible for the trait value differences between lines. For both objective traits examined, exome QTL-seq identified the monogenic Mendelian locus and associated QTLs. These findings were validated using conventional mapping approaches. Conclusions Exome QTL-seq broadens the utility of NGS-based gene/QTL mapping in organisms with large genomes.

Barley ( Hordeum vulgare ) mildew locus o ( mlo ) mutants exhibit strong resistance to penetration by the powdery mildew fungus Blumeria graminis f. sp. hordei . MLO, a seven-transmembrane protein localized at the plasma membrane is thought to be involved in intracellular calcium signaling. However, its molecular function and the mechanism by which mlo mutations confer resistance to penetration by the fungus remain poorly understood. A large number of mlo alleles with different amino acid substitutions at each intracellular loop have been found in various cultivars. However, it is difficult to analyze how each amino acid is involved in penetration resistance by comparing these cultivars because they differ substantially in their genetic background and in the presence or absence of resistance genes recognizing avirulence factors from the pathogen. In this study, we used a CRISPR/Cas9-mediated genetic modification system to generate mlo mutants in the transformable cultivar Golden Promise to enable complementation experiments with the aim of elucidating the molecular function of MLO. An mlo mutant with a thymine insertion in the second exon and penetration resistance to B . graminis f. sp. hordei was obtained. Susceptibility was restored in cells in which the MLO-mCherry gene was introduced using particle bombardment, indicating that this mlo mutant could be a useful genetic tool for complementation experiments using transgenes expressing a variety of mlo alleles.

The energy in cellulosic biomass largely resides in plant cell walls. Cellulosic biomass is more difficult than starch to break down into sugars because of the presence of lignin and the complex structure of cell walls. Transgenic down-regulation of major lignin genes led to reduced lignin content, increased dry matter degradability, and improved accessibility of cellulases for cellulose degradation. This review provides background information on lignin biosynthesis and focuses on genetic manipulation of lignin genes in important monocot species as well as the dicot potential biofuel crop alfalfa. Reduction of lignin in biofuel crops by genetic engineering is likely one of the most effective ways of reducing costs associated with pretreatment and hydrolysis of cellulosic feedstocks, although some potential fitness issues should also be addressed.

Loss-of-function mutation of the MILDEW RESISTANCE LOCUS O ( Mlo ) gene confers durable and broad-spectrum resistance to powdery mildew fungi in various plants, including barley. In combination with the intracellular nucleotide-binding domain and leucine-rich repeat receptor ( NLR ) genes, which confer the race-specific resistance, the mlo alleles have long been used in barley breeding as genetic resources that confer robust non-race-specific resistance. However, a Japanese Blumeria graminis f. sp. hordei isolate, RACE1, has been reported to have the potential to overcome partially the mlo -mediated penetration resistance, although this is yet uncertain because the putative effects of NLR genes in the tested accessions have not been ruled out. In this study, we examined the reproducibility of the earlier report and found that the infectious ability of RACE1, which partially overcomes the mlo -mediated resistance, is only exerted in the absence of NLR genes recognizing RACE1. Furthermore, using the transient-induced gene silencing technique, we demonstrated that RACE1 can partially overcome the resistance in the host cells with suppressed MLO expression but not in plants possessing the null mutant allele mlo -5.

Limitations for the application of genome editing technologies on elite wheat ( Triticum aestivum L.) varieties are mainly due to the dependency on in vitro culture and regeneration capabilities. Recently, we developed an in planta particle bombardment (iPB) method which has increased process efficiency since no culture steps are required to create stably genome-edited wheat plants. Here, we report the application of the iPB method to commercially relevant Japanese elite wheat varieties. The biolistic delivery of gold particles coated with plasmids expressing CRISPR/Cas9 components designed to target TaQsd1 were bombarded into the embryos of imbibed seeds with their shoot apical meristem (SAM) exposed. Mutations in the target gene were subsequently analyzed within flag leaf tissue by using cleaved amplified polymorphic sequence (CAPS) analysis. A total of 9/358 (2.51%) of the bombarded plants (cv. “Haruyokoi,” spring type) carried mutant alleles in the tissue. Due to the chimeric nature of the T0 plants, only six of them were inherited to the next (T1) generation. Genotypic analysis of the T2 plants revealed a single triple-recessive homozygous mutant of the TaQsd1 gene. Compared to wild type, the homozygous mutant exhibited a 7 days delay in the time required for 50% seed germination. The iPB method was also applied to two elite winter cultivars, “Yumechikara” and “Kitanokaori,” which resulted in successful genome editing at slightly lower efficiencies as compared to “Haruyokoi.” Taken together, this report demonstrates that the in planta genome editing method through SAM bombardment can be applicable to elite wheat varieties that are otherwise reluctant to callus culture.

It is necessary to overcome recalcitrance of the biomass to saccharification (sugar release) to make switchgrass (Panicum virgatum) economically viable as a feedstock for liquid biofuels. Lignin content correlates negatively with sugar release efficiency in switchgrass, but selecting the right gene candidates for engineering lignin biosynthesis in this tetraploid outcrossing species is not straightforward. To assist this endeavor, we have used an inducible switchgrass cell suspension system for studying lignin biosynthesis in response to exogenous brassinolide. By applying a combination of protein sequence phylogeny with whole-genome microarray analyses of induced cell cultures and developing stem internode sections, we have generated a list of candidate monolignol biosynthetic genes for switchgrass. Several genes that were strongly supported through our bioinformatics analysis as involved in lignin biosynthesis were confirmed by gene silencing studies, in which lignin levels were reduced as a result of targeting a single gene. However, candidate genes encoding enzymes involved in the early steps of the currently accepted monolignol biosynthesis pathway in dicots may have functionally redundant paralogues in switchgrass and therefore require further evaluation. This work provides a blueprint and resources for the systematic genome-wide study of the monolignol pathway in switchgrass, as well as other C4 monocot species.