Model Grass Species Research Articles

Development of SRAP markers to evaluate the genetic variability in Brachypodium distachyon complex

ABSTRACT Brachypodium distachyon has been accepted as a model grass species for genetics and molecular genomics in cereals since 2001. However, the genetic variability present in Brachypodium spp. continue being the aim of many research. Therefore, the aim of this work was to analyze the genetic variability present in different Brachypodium spp. from different countries and to find different open reading frames (ORFs) among them by using sequence-related amplified polymorphism (SRAP) markers. In our study, a total of 12 Brachypodium spp. accessions were selected from different countries. By using SRAP markers, the genetic variability was evaluated and some fragments were sequenced to obtain differential ORF information among genotypes. SRAP molecular markers revealed high variability among accessions being USA the accession that showed the most variability at the DNA level. The analysis of sequenced SRAP fragments from monomorphic and polymorphic fragments as well as those obtained from new primer combinations targeted coding regions into B. distachyon available genome. SRAP molecular marker showed not only to be an excellent tool for Brachypodium variability studies but also to provide additional information about differential amplification of open reading frames.

Brachypodium: 20 years as a grass biology model system; the way forward?

It has been 20 years since Brachypodium distachyon was suggested as a model grass species, but ongoing research now encompasses the entire genus. Extensive Brachypodium genome sequencing programmes have provided resources to explore the determinants and drivers of population diversity. This has been accompanied by cytomolecular studies to make Brachypodium a platform to investigate speciation, polyploidisation, perenniality, and various aspects of chromosome and interphase nucleus organisation. The value of Brachypodium as a functional genomic platform has been underscored by the identification of key genes for development, biotic and abiotic stress, and cell wall structure and function. While Brachypodium is relevant to the biofuel industry, its impact goes far beyond that as an intriguing model to study climate change and combinatorial stress.

Targeted and Untargeted Metabolomic Analyses Reveal Organ Specificity of Specialized Metabolites in the Model Grass Brachypodium distachyon.

Brachypodium distachyon, because of its fully sequenced genome, is frequently used as a model grass species. However, its metabolome, which constitutes an indispensable element of complex biological systems, remains poorly characterized. In this study, we conducted comprehensive, liquid chromatography-mass spectrometry (LC-MS)-based metabolomic examination of roots, leaves and spikes of Brachypodium Bd21 and Bd3-1 lines. Our pathway enrichment analysis emphasised the accumulation of specialized metabolites representing the flavonoid biosynthetic pathway in parallel with processes related to nucleotide, sugar and amino acid metabolism. Similarities in metabolite profiles between both lines were relatively high in roots and leaves while spikes showed higher metabolic variance within both accessions. In roots, differences between Bd21 and Bd3-1 lines were manifested primarily in diterpenoid metabolism, while differences within spikes and leaves concerned nucleotide metabolism and nitrogen management. Additionally, sulphate-containing metabolites differentiated Bd21 and Bd3-1 lines in spikes. Structural analysis based on MS fragmentation spectra enabled identification of 93 specialized metabolites. Among them phenylpropanoids and flavonoids derivatives were mainly determined. As compared with closely related barley and wheat species, metabolic profile of Brachypodium is characterized with presence of threonate derivatives of hydroxycinnamic acids.

A novel plant-fungal association reveals fundamental sRNA and gene expression reprogramming at the onset of symbiosis

Abstract Background Beneficial associations between plants and microbes are widespread in nature and have been studied extensively in the microbial-dominant environment of the rhizosphere. Such associations are highly advantageous for the organisms involved, benefiting soil microbes by providing them access to plant metabolites, while plant growth and development are enhanced through the promotion of nutrient uptake and/or protection against (a)biotic stresses. While the establishment and maintenance of mutualistic associations have been shown to require genetic and epigenetic reprogramming, as well as an exchange of effector molecules between microbes and plants, whether short RNAs are able to effect such changes is currently unknown. Here, we established an interaction between the model grass species Brachypodium distachyon (Bd, Pooideae) and the beneficial fungal root endophyte Serendipita indica (Si, syn. Piriformospora indica, Sebacinales) to elucidate RNA interference-based regulatory changes in gene expression and small (s)RNA profiles that occurred during establishment of a Sebacinalean symbiosis. Results Colonization of Bd roots with Si resulted in higher grain yield, confirming the mutualistic character of this interaction. Resequencing of the Si genome using the Oxford Nanopore technique, followed by de novo assembly yielded in 57 contigs and 9441 predicted genes, including putative members of several families involved in sRNA production. Transcriptome analysis at an early stage of the mutualistic interaction identified 2963 differentially expressed genes (DEG) in Si and 317 in Bd line 21-3. The fungal DEGs were largely associated with carbohydrate metabolism, cell wall degradation, and nutrient uptake, while plant DEGs indicated modulation of (a)biotic stress responses and defense pathways. Additionally, 10% of the upregulated fungal DEGs encode candidate protein effectors, including six DELD proteins typical for Sebacinales. Analysis of the global changes in the sRNA profiles of both associated organisms revealed several putative endogenous plant sRNAs expressed during colonization belonging to known micro (mi)RNA families involved in growth and developmental regulation. Among Bd- and Si-generated sRNAs with putative functions in the interacting organism, we identified transcripts for proteins involved in circadian clock and flowering regulation as well as immunity as potential targets of fungal sRNAs, reflecting the beneficial activity of Si. Conclusions We detected beneficial effects of Si colonization on Bd growth and development, and established a novel plant-mutualist interaction model between these organisms. Together, the changes in gene expression and identification of interaction-induced sRNAs in both organisms support sRNA-based regulation of defense responses and plant development in Bd, as well as nutrient acquisition and cell growth in Si. Our data suggests that a Sebacinalean symbiosis involves reciprocal sRNA targeting of genes during the interaction.

Grasviq: an image analysis framework for automatically quantifying vein number and morphology in grass leaves.

Beyond facilitating transport and providing mechanical support to the leaf, veins have important roles in the performance and productivity of plants and the ecosystem. In recent decades, computational image analysis has accelerated the extraction and quantification of vein traits, benefiting fields of research from agriculture to climatology. However, most of the existing leaf vein image analysis programs have been developed for the reticulate venation found in dicots. Despite the agroeconomic importance of cereal grass crops, like Oryza sativa (rice) and Zea mays (maize), a dedicated image analysis program for the parallel venation found in monocots has yet to be developed. To address the need for an image-based vein phenotyping tool for model and agronomic grass species, we developed the grass vein image quantification (grasviq) framework. Designed specifically for parallel venation, this framework automatically segments and quantifies vein patterns from images of cleared leaf pieces using classical computer vision techniques. Using image data sets from maize inbred lines and auxin biosynthesis and transport mutants in maize, we demonstrate the utility of grasviq for quantifying important vein traits, including vein density, vein width and interveinal distance. Furthermore, we show that the framework can resolve quantitative differences and identify vein patterning defects, which is advantageous for genetic experiments and mutant screens. We report that grasviq can perform high-throughput vein quantification, with precision on a par with that of manual quantification. Therefore, we envision that grasviq will be adopted for vein phenomics in maize and other grass species.

Heterologous Overexpression of Arabidopsis cel1 Enhances Grain Yield, Biomass and Early Maturity in Setaria viridis.

Heterologous overexpression of Arabidopsis cellulase 1 (Atcel1) results in enhanced yield, early maturity, and increased biomass in dicotyledonous species like poplar and eucalyptus but has not been demonstrated in monocots. We produced transgenic Setaria viridis accession A10.1 plants overexpressing a monocotyledonous codon optimized (MCO) Atcel1. Agronomic characterization of the transgenic events showed that heterologous overexpression of MCOAtcel1 caused enhanced grain yield, shoot biomass, and accelerated maturation rate in the model grass species S. viridis under growth chamber conditions. The agronomic trait differences observed were consistent with previous reports in dicots but are here described in a monocot species and associated with increased seed yield. Overexpression of Atcel1 in S. viridis was shown to increase the number of panicles and seeds by 24–30%, enhance overall grain yield by up to 26%, and lead to a shoot dry biomass increase of 16–19%. Overexpression also reduced time to plant maturation and senescence by 12.5%. Our findings in S. viridis suggest that manipulation of Atcel1 has potential for developing early-maturing and higher-yielding monocotyledonous biomass crops suitable for climate-smart agriculture.

Dynamic cytokinin signaling and function of auxin in cytokinin responsive domains during rice crown root development

We reveal the onset and dynamic tissue-specific cytokinin signaling domains and functional importance of auxin in the auxin-cytokinin interaction domains in shaping root architecture in the economically important rice plant. Plant hormones such as auxin and cytokinin are central regulators of root organogenesis. Typical in the grass species, the root system in rice is primarily composed of post-embryonic adventitious/crown roots (ARs/CRs). Antagonistic auxin-cytokinin activities mutually balance each other to ensure proper root development. Cytokinin has been shown to inhibit crown root initiation in rice; albeit, the responsive domains remain elusive during the initiation and outgrowth of crown root primordia (CRP). Here, we show the cytokinin response domains during various stages of CRP development. RNA-RNA in situ hybridization and protein immunohistochemistry studies of the reporter gene expressed under the cytokinin responsive synthetic promoter revealed detailed spatio-temporal cytokinin signaling domains in the developing CRP. Furthermore, rice lines genetically depleted for endogenous auxin in the cytokinin responsive domains provided insight into the functional importance of auxin signaling during crown root development. Thus, our study demonstrates the onset and dynamic tissue-specific cytokinin response and functional significance of auxin-cytokinin interaction during root architecture formation in rice, a model grass species.

Genetic and Methylome Variation in Turkish Brachypodium Distachyon Accessions Differentiate Two Geographically Distinct Subpopulations.

Brachypodium distachyon (Brachypodium) is a non-domesticated model grass species that can be used to test if variation in genetic sequence or methylation are linked to environmental differences. To assess this, we collected seeds from 12 sites within five climatically distinct regions of Turkey. Seeds from each region were grown under standardized growth conditions in the UK to preserve methylated sequence variation. At six weeks following germination, leaves were sampled and assessed for genomic and DNA methylation variation. In a follow-up experiment, phenomic approaches were used to describe plant growth and drought responses. Genome sequencing and population structure analysis suggested three ancestral clusters across the Mediterranean, two of which were geographically separated in Turkey into coastal and central subpopulations. Phenotypic analyses showed that the coastal subpopulation tended to exhibit relatively delayed flowering and the central, increased drought tolerance as indicated by reduced yellowing. Genome-wide methylation analyses in GpC, CHG and CHH contexts also showed variation which aligned with the separation into coastal and central subpopulations. The climate niche modelling of both subpopulations showed a significant influence from the “Precipitation in the Driest Quarter” on the central subpopulation and “Temperature of the Coldest Month” on the coastal subpopulation. Our work demonstrates genetic diversity and variation in DNA methylation in Turkish accessions of Brachypodium that may be associated with climate variables and the molecular basis of which will feature in ongoing analyses.

A reformulation of Murashige and Skoog medium (WPBS medium) improves embryogenesis, morphogenesis and transformation efficiency in temperate and tropical grasses and cereals

Slow callus growth is a barrier to efficient genetic transformation in some gramineous species. A reformulation of Murashige and Skoog (MS) medium, with additional magnesium sulphate, potassium phosphate, copper sulphate, proline and glutamine, termed WPBS medium, has been developed which improves all aspects of in vitro culture when compared with MS based media. Embryogenic callus could be produced more rapidly from responsive genotypes of sixteen cereal, forage, model and energy grass species, whether using embryos, shoot tips or proliferated meristems as explants. Three species were not transformed due to contamination or unsuitable explant, but thirteen species were transformed using an identical Agrobacterium-mediated transformation, selection and regeneration protocol, including Avena sativa and Oryza sativa. Readily transformable species such as Lolium perenne, Brachypodium distachyon and Festuca arundinacea and recalcitrant species such as Lolium temulentum and Miscanthus sinensis were reliably transformed, while two new species Phalaris arundinacea and viviparous Deschampsia cespitosa were transformed at the first attempt. It is hoped that the use of WPBS media and this general transformation protocol may help to improve the efficiency of grass and cereal transformation.

In-Situ Metabolomic Analysis of Setaria viridis Roots Colonized by Beneficial Endophytic Bacteria.

Over the past decades, crop yields have risen in parallel with increasing use of fossil fuel-derived nitrogen (N) fertilizers but with concomitant negative impacts on climate and water resources. There is a need for more sustainable agricultural practices, and biological nitrogen fixation (BNF) could be part of the solution. A variety of nitrogen-fixing, epiphytic, and endophytic plant growth-promoting bacteria (PGPB) are known to stimulate plant growth. However, compared with the rhizobium-legume symbiosis, little mechanistic information is available as to how PGPB affect plant metabolism. Therefore, we investigated the metabolic changes in roots of the model grass species Setaria viridis upon endophytic colonization by Herbaspirillum seropedicae SmR1 (fix+) or a fix- mutant strain (SmR54) compared with uninoculated roots. Endophytic colonization of the root is highly localized and, hence, analysis of whole-root segments dilutes the metabolic signature of those few cells impacted by the bacteria. Therefore, we utilized in-situ laser ablation electrospray ionization mass spectrometry to sample only those root segments at or adjacent to the sites of bacterial colonization. Metabolites involved in purine, zeatin, and riboflavin pathways were significantly more abundant in inoculated plants, while metabolites indicative of nitrogen, starch, and sucrose metabolism were reduced in roots inoculated with the fix- strain or uninoculated, presumably due to N limitation. Interestingly, compounds, involved in indole-alkaloid biosynthesis were more abundant in the roots colonized by the fix- strain, perhaps reflecting a plant defense response.

Physiological acclimation of a grass species occurs during sustained but not repeated drought events

Climate change is predicted to result in more variable and extreme precipitation regimes, with potentially longer periods between precipitation events. However, the effects of multiple, sequential drought events on ecosystems and potential acclimation, particularly in grasslands, is poorly understood. In this study, we tested if two simulated drought regimes, (i) sustained drought (soil moisture content reduced from 75 to 25 % field capacity over 14 days and maintained for 29 days) and (ii) repeated drought (two cycles of a reduction to 25 % field capacity, reached after 22 days and 15 days, separated by a rewatering event to 75 % field capacity for 5 days), differentially influence fourteen key physiological responses in the model grass species (Brachypodium distachyon). In addition, we aimed to determine whether plants may acclimate differently to sustained and/or repeated drought events. B. distachyon exhibited plasticity in response to drought, exhibiting two distinct drought resistance responses to the two watering regimes. Plants exposed to sustained drought exhibited early reductions in photosynthesis, stomatal conductance and water potential (43 %, 42 % and 37 %, respectively). However, plants showed physiological acclimation and recovery in these parameters after an additional 3–21 days, but had a ∼50 % lower shoot biomass than well-watered plants. These data demonstrate a conservative, albeit plastic, response to soil water deficit. In contrast, plants exposed to repeated drought responded opportunistically whereby greater fluctuations in photosynthesis, stomatal conductance and water potential (reductions of up to 62 %, 71 % and 90 %, respectively) were closely related to drought and rewatering events. Since plants exposed to repeated drought had a greater reduction in water potential and faster reductions in relative photosynthesis in the second drought event compared to the initial drought event, this may indicate a lack of acclimation under this drought regime. Our results provide evidence for differential responses of B. distachyon to drought and rewatering events, which are dependent on the drought regime (sustained or repeated) and must be carefully considered when interpreting experimental data.

Further Elucidation of the Argonaute and Dicer Protein Families in the Model Grass Species Brachypodium distachyon.

RNA interference (RNAi) is a biological process in which small RNAs regulate gene silencing at the transcriptional or posttranscriptional level. The trigger for gene silencing is double-stranded RNA generated from an endogenous genomic locus or a foreign source, such as a transgene or virus. In addition to regulating endogenous gene expression, RNAi provides the mechanistic basis for small RNA-mediated communication between plant hosts and interacting pathogenic microbes, known as cross-kingdom RNAi. Two core protein components, Argonaute (AGO) and Dicer (DCL), are central to the RNAi machinery of eukaryotes. Plants encode for several copies of AGO and DCL genes; in Arabidopsis thaliana, the AGO protein family contains 10 members, and the DCL family contains four. Little is known about the conservation and specific roles of these proteins in monocotyledonous plants, which account for the most important food staples. Here, we utilized in silico tools to investigate the structure and related functions of AGO and DCL proteins from the model grass Brachypodium distachyon. Based on the presence of characteristic domains, 16 BdAGO- and 6 BdDCL-predicted proteins were identified. Phylogenetic analysis showed that both protein families were expanded in Brachypodium as compared with Arabidopsis. For BdDCL proteins, both plant species contain a single copy of DCL1 and DCL4; however, Brachypodium contains two copies each of DCL2 and DCL3. Members of the BdAGO family were placed in all three functional clades of AGO proteins previously described in Arabidopsis. The greatest expansion occurred in the AtAGO1/5/10 clade, which contains nine BdAGOs (BdAGO5/6/7/9/10/11/12/15/16). The catalytic tetrad of the AGO P-element-induced wimpy testis domain (PIWI), which is required for endonuclease activity, is conserved in most BdAGOs, with the exception of BdAGO1, which lacks the last D/H residue. Three-dimensional modeling of BdAGO proteins using tertiary structure prediction software supported the phylogenetic classification. We also predicted a provisional interactome network for BdAGOs, their localization within the cell, and organ/tissue-specific expression. Exploring the specifics of RNAi machinery proteins in a model grass species can serve as a proxy for agronomically important cereals such as barley and wheat, where the development of RNAi-based plant protection strategies is of great interest.

Partial defoliation of Brachypodium distachyon plants grown in petri dishes under low light increases P and other nutrient levels concomitantly with transcriptional changes in the roots.

BackgroundThere have been few studies on the partial defoliation response of grass. It has been unclear how partial defoliation may affect roots at the levels of nutrient accumulation and transcriptional regulation. Hereby we report a comprehensive investigation on molecular impacts of partial defoliation by using a model grass species, Brachypodium distachyon.ResultsOur Inductively Coupled Plasma Mass Spectrometry analyses of B. distachyon revealed shoot- and root-specific accumulation patterns of a group of macronutrients including potassium (K), Phosphorus (P), Calcium (Ca), Magnesium (Mg), and micronutrients including Sodium (Na), iron (Fe), and Manganese (Mn). Meanwhile, our genome-wide profiling of gene expression patterns depicts transcriptional impacts on B. distachyon roots by cutting the aerial portion. The RNAseq analyses identified a total of 1,268 differentially expressed genes in B. distachyon with partial defoliation treatment. Our comprehensive analyses by means of multiple approaches, including Gene Ontology, InterPro and Pfam protein classification, KEGG pathways, and Plant TFDB, jointly highlight the involvement of hormone-mediated wounding response, primary and secondary metabolites, and ion homeostasis, in B. distachyon after the partial defoliation treatment. In addition, evidence is provided that roots respond to partial defoliation by modifying nutrient uptake and rhizosphere acidification rate, indicating that an alteration of the root/soil interaction occurs in response to this practice.ConclusionsThis study reveals how partial defoliation alters ion accumulation levels in shoots and roots, as well as partial defoliation-induced transcriptional reprogramming on a whole-genome scale, thereby providing insight into the molecular mechanisms underlying the recovery process of grass after partial defoliation.

Transcriptome analysis of the model grass Lolium temulentum exposed to green leaf volatiles

BackgroundForage and turf grasses are routinely cut and grazed upon throughout their lifecycle. When grasses are cut or damaged, they rapidly release a volatile chemical cocktail called green leaf volatiles (GLV). Previously we have shown that mechanical wounding or exposure to GLV released from cut grass, activated a Lt 46 kDa mitogen-activated protein kinase (MAPK) within 3 min and a 44 kDa MAPK within 15–20 min in the model grass species Lolium temulentum (Lt). Currently very little is known concerning the perception, signaling or molecular responses associated with wound stress in grasses. Since GLV are released during wounding, we wanted to investigate what genes and signaling pathways would be induced in undamaged plants exposed to GLV.ResultsRNA-Seq generated transcriptome of Lolium plants exposed to GLV identified 4308 up- and 2794 down-regulated distinct differentially-expressed sequences (DES). Gene Ontology analysis revealed a strong emphasis on signaling, response to stimulus and stress related categories. Transcription factors and kinases comprise over 13% of the total DES found in the up-regulated dataset. The analysis showed a strong initial burst within the first hour of GLV exposure with over 60% of the up-regulated DES being induced. Specifically sequences annotated for enzymes involved in the biosynthesis of jasmonic acid and other plant hormones, mitogen-activated protein kinases and WRKY transcription factors were identified. Interestingly, eleven DES for ferric reductase oxidase, an enzyme involved in iron uptake and transport, were exclusively found in the down-regulated dataset. Twelve DES of interest were selected for qRT-PCR analysis; all displayed a rapid induction one hour after GLV exposure and were also strongly induced by mechanical wounding.ConclusionThe information gained from the analysis of this transcriptome and previous studies suggests that GLV released from cut grasses transiently primes an undamaged plant’s wound stress pathways for potential oncoming damage, and may have a dual role for inter- as well as intra-plant signaling.

OsMYB108 loss-of-function enriches p-coumaroylated and tricin lignin units in rice cell walls.

Breeding approaches to enrich lignins in biomass could be beneficial to improving the biorefinery process because lignins increase biomass heating value and represent a potent source of valuable aromatic chemicals. However, despite the fact that grasses are promising lignocellulose feedstocks, limited information is yet available for molecular-breeding approaches to upregulate lignin biosynthesis in grass species. In this study, we generated lignin-enriched transgenic rice (Oryza sativa), a model grass species, via targeted mutagenesis of the transcriptional repressor OsMYB108 using CRISPR/Cas9-mediated genome editing. The OsMYB108-knockout rice mutants displayed increased expressions of lignin biosynthetic genes and enhanced lignin deposition in culm cell walls. Chemical and two-dimensional nuclear magnetic resonance (NMR) analyses revealed that the mutant cell walls were preferentially enriched in γ-p-coumaroylated and tricin lignin units, both of which are typical and unique components in grass lignins. NMR analysis also showed that the relative abundances of major lignin linkage types were altered in the OsMYB108 mutants.

Genome size variation among natural populations of Brachypodium distachyon and B. hybridum collected from different regions of Turkey

Brachypodium distachyon (L.) P.Beauv. is a model grass species that is useful for studying temperate cereal, forage, and energy crops. In this study we aimed to determine the genome size and species identity in the collections of the B. distachyon complex that included samples from 56 different locations in Turkey, applying chromosome counting and nuclear genome size evaluation by flow cytometry method (FCM). Moreover, the study examined whether diverse geographical conditions at the collecting sites influence the genome size. Chromosomal analysis revealed that the number of B. distachyon (x = 5, 2n = 10) genotypes was 48 and the number of B. hybridum (x = 5 + 10, 2n = 30) ones was 11. No genotypes of B. stacei were found. FCM analyses revealed that the minimum and maximum genome size of B. distachyon was 0.732 and 0.752 pg2C?1, respectively. The mean genome size of B. distachyon was 0.743 pg2C?1. The mean genome size of B. hybridum was 1.431 pg2C?1 with the minimum 1.417 pg2C?1 and maximum 1.451 pg2C?1. Intraspecific variation in the genome size was identified for both species. It was determined that geographical origin (localization, altitude) had a statistically significant effect on genome size in B. distachyon. In addition, a negative correlation was found between altitude and genome size in this species. On the other hand, localization and altitude did not have any statistically significant effect on genome size in B. hybridum.

Identification and characterization of genes encoding the nuclear envelope LINC complex in the monocot speciesZea mays

The linker of nucleoskeleton to cytoskeleton (LINC) complex is an essential multi-protein structure spanning the nuclear envelope. It connects the cytoplasm to the nucleoplasm, functions to maintain nuclear shape and architecture and regulates chromosome dynamics during cell division. Knowledge of LINC complex composition and function in the plant kingdom is primarily limited to Arabidopsis, but critically missing from the evolutionarily distant monocots, which include grasses, the most important agronomic crops worldwide. To fill this knowledge gap, we identified and characterized 22 maize genes, including a new grass-specific KASH gene family. By using bioinformatic, biochemical and cell biological approaches, we provide evidence that representative KASH candidates localize to the nuclear periphery and interact with Zea mays (Zm)SUN2 in vivo FRAP experiments using domain deletion constructs verified that this SUN-KASH interaction was dependent on the SUN but not the coiled-coil domain of ZmSUN2. A summary working model is proposed for the entire maize LINC complex encoded by conserved and divergent gene families. These findings expand our knowledge of the plant nuclear envelope in a model grass species, with implications for both basic and applied cellular research.This article has an associated First Person interview with the first author of the paper.

Lignin characterization of rice CONIFERALDEHYDE 5-HYDROXYLASE loss-of-function mutants generated with the CRISPR/Cas9 system.

The aromatic composition of lignin is an important trait that greatly affects the usability of lignocellulosic biomass. We previously identified a rice (Oryza sativa) gene encoding coniferaldehyde 5-hydroxylase (OsCAld5H1), which was effective in modulating syringyl (S)/guaiacyl (G) lignin composition ratio in rice, a model grass species. Previously characterized OsCAld5H1-knockdown rice lines, which were produced via an RNA-interference approach, showed augmented G lignin units yet contained considerable amounts of residual S lignin units. In this study, to further investigate the effect of suppression of OsCAld5H1 on rice lignin structure, we generated loss-of-function mutants of OsCAld5H1 using the CRISPR/Cas9-mediated genome editing system. Homozygous OsCAld5H1-knockout lines harboring anticipated frame-shift mutations in OsCAld5H1 were successfully obtained. A series of wet-chemical and two-dimensional NMR analyses on cell walls demonstrated that although lignins in the mutant were predictably enriched in G units all the tested mutant lines produced considerable numbers of S units. Intriguingly, lignin γ-p-coumaroylation analysis by the derivatization followed by reductive cleavage method revealed that enrichment of G units in lignins of the mutants was limited to the non-γ-p-coumaroylated units, whereas grass-specific γ-p-coumaroylated lignin units were almost unaffected. Gene expression analysis indicated that no homologous genes of OsCAld5H1 were overexpressed in the mutants. These data suggested that CAld5H is mainly involved in the production of non-γ-p-coumaroylated S lignin units, common in both eudicots and grasses, but not in the production of grass-specific γ-p-coumaroylated S units in rice.

Regulation of cell wall development in Brachypodium distachyon in vitro as affected by cytokinin and gas exchange

Brachypodium distachyon is successfully used as a model grass species. The present study used molecular and chemical approaches to investigate the effects of the addition of the cytokinin 6-benzyladenine (BA) and two levels of gas exchange on the transcriptional regulation of lignin biosynthesis in B. distachyon. It was observed that the control of lignin biosynthesis is affected by both BA and the in vitro gas exchange rate, by sealing the vial with absence or presence of membranes. These observations are supported by anatomical and antioxidant enzyme activity analyses. However, the hyperhydricity caused by BA did not affect cell wall properties in B. distachyon. Interestingly, the CCR1 gene presented 70-fold increase in expression under the effect of BA and without membranes in addition to a significant increase in the peroxidases activity and total lignin content. Treatments with gas exchange showed higher qualitative accumulation of lignin S/G. Lignin pathway genes were transcriptionally coordinated according to the imposed treatment with apparent co-expression. Low expression of these genes was observed in the sealing treatment without membranes, while the opposite was verified in the condition of sealing with two membranes. CESA4 and CESA7 genes responded positively to gas exchange, without altering the cell wall cellulose/lignin ratio in B. distachyon. These results contribute to a better understanding of how cell wall-related gene expression is regulated by exogenous factors, as well as the many possibilities of cell wall manipulation due to its plasticity in adaptive responses.

Tissue Culture (Somatic Embryogenesis)-Induced Tnt1 Retrotransposon-Based Mutagenesis in Brachypodium distachyon.

Brachypodium distachyon is a model grass species for economically important cereal crops. Efforts are in progress to develop useful functional genomic resources in Brachypodium. A tobacco retrotransposon, Tnt1, has been used successfully in recent past to generate insertional mutagenesis in several dicot plant species. Tnt1 retrotransposon replicates, transposes, and inserts at multiple random genomic locations in the plant genome. Transposition occurs only during somatic embryogenesis but not during seed transmission. We developed Brachypodium transgenic plants that can express the Tnt1 element. Here, we describe an efficient tissue culture-based approach to generate Tnt1 insertional mutant population using transgenic Brachypodium line expressing the Tnt1 retrotransposon.