Grass xylan structural variation suggests functional specialization and distinctive interaction with cellulose and lignin.

Xylan is the most abundant non-cellulosic polysaccharide in grass cell walls, and it has important structural roles. The name glucuronoarabinoxylan (GAX) is used to describe this variable hemicellulose. It has a linear backbone of ß-1,4-xylose (Xyl) residues that may be substituted with α-1,2-linked (4-O-methyl)-glucuronic acid (GlcA), α-1,3-linked arabinofuranose (Araf), and sometimes acetylation at the O-2 and/or O-3 positions. The role of these substitutions remains unclear, although there is increasing evidence that they affect the way xylan interacts with other cell wall components, particularly cellulose and lignin. Here, we used substitution-dependent endo-xylanase enzymes to investigate the variability of xylan substitution in grass culm cell walls. We show that there are at least three different types of xylan: (i) an arabinoxylan with evenly distributed Araf substitutions without GlcA (AXe); (ii) a glucuronoarabinoxylan with clustered GlcA modifications (GAXc); and (iii) a highly substituted glucuronoarabinoxylan (hsGAX). Immunolocalization of AXe and GAXc in Brachypodium distachyon culms revealed that these xylan types are not restricted to a few cell types but are instead widely detected in Brachypodium cell walls. We hypothesize that there are functionally specialized xylan types within the grass cell wall. The even substitutions of AXe may permit folding and binding on the surface of cellulose fibrils, whereas the more complex substitutions of the other xylans may support a role in the matrix and interaction with other cell wall components.

MsHDZ23, a Novel Miscanthus HD-ZIP Transcription Factor, Participates in Tolerance to Multiple Abiotic Stresses.

The homeodomain-leucine zipper (HD-ZIP) transcription factors, representing one of the largest plant-specific superfamilies, play important roles in the response to various abiotic stresses. However, the functional roles of HD-ZIPs in abiotic stress tolerance and the underlying mechanisms remain relatively limited in Miscanthus sinensis. In this study, we isolated an HD-ZIP TF gene, MsHDZ23, from Miscanthus and ectopically expressed it in Arabidopsis. Transcriptome and promoter analyses revealed that MsHDZ23 responded to salt, alkali, and drought treatments. The overexpression (OE) of MsHDZ23 in Arabidopsis conferred higher tolerance to salt and alkali stresses compared to wild-type (WT) plants. Moreover, MsHDZ23 was able to restore the hb7 mutant, the ortholog of MsHDZ23 in Arabidopsis, to the WT phenotype. Furthermore, MsHDZ23-OE lines exhibited significantly enhanced drought stress tolerance, as evidenced by higher survival rates and lower water loss rates compared to WT. The improved drought tolerance may be attributed to the significantly smaller stomatal aperture in MsHDZ23-OE lines compared to WT. Furthermore, the accumulation of the malondialdehyde (MDA) under abiotic stresses was significantly decreased, accompanied by dramatically enhanced activities in several antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in the transgenic plants. Collectively, these results demonstrate that MsHDZ23 functions as a multifunctional transcription factor in enhancing plant resistance to abiotic stresses.

Genome-Wide Characterization and Expression Profiling of the GRAS Gene Family in Salt and Alkali Stresses in Miscanthus sinensis.

The GRAS family genes encode plant-specific transcription factors that play important roles in a diverse range of developmental processes and abiotic stress responses. However, the information of GRAS gene family in the bioenergy crop Miscanthus has not been available. Here, we report the genome-wide identification of GRAS gene family in Micanthus sinensis. A total of 123 MsGRAS genes were identified, which were divided into ten subfamilies based on the phylogenetic analysis. The co-linearity analysis revealed that 59 MsGRAS genes experienced segmental duplication, forming 35 paralogous pairs. The expression of six MsGRAS genes in responding to salt, alkali, and mixed salt-alkali stresses was analyzed by transcriptome and real-time quantitative PCR (RT-qPCR) assays. Furthermore, the role of MsGRAS60 in salt and alkali stress response was characterized in transgenic Arabidopsis. The MsGRAS60 overexpression lines exhibited hyposensitivity to abscisic acid (ABA) treatment and resulted in compromised tolerance to salt and alkali stresses, suggesting that MsGRAS60 is a negative regulator of salt and alkali tolerance via an ABA-dependent signaling pathway. The salt and alkali stress-inducible MsGRAS genes identified serve as candidates for the improvement of abiotic stress tolerance in Miscanthus.

Unveiling the transcriptomic complexity of Miscanthus sinensis using a combination of PacBio long read- and Illumina short read sequencing platforms.

BACKGROUND: Miscanthus sinensis Andersson is a perennial grass that exhibits remarkable lignocellulose characteristics suitable for sustainable bioenergy production. However, knowledge of the genetic resources of this species is relatively limited, which considerably hampers further work on its biology and genetic improvement. RESULTS: In this study, through analyzing the transcriptome of mixed samples of leaves and stems using the latest PacBio Iso-Seq sequencing technology combined with Illumina HiSeq, we report the first full-length transcriptome dataset of M. sinensis with a total of 58.21 Gb clean data. An average of 15.75 Gb clean reads of each sample were obtained from the PacBio Iso-Seq system, which doubled the data size (6.68 Gb) obtained from the Illumina HiSeq platform. The integrated analyses of PacBio- and Illumina-based transcriptomic data uncovered 408,801 non-redundant transcripts with an average length of 1,685 bp. Of those, 189,406 transcripts were commonly identified by both methods, 169,149 transcripts with an average length of 619 bp were uniquely identified by Illumina HiSeq, and 51,246 transcripts with an average length of 2,535 bp were uniquely identified by PacBio Iso-Seq. Approximately 96 % of the final combined transcripts were mapped back to the Miscanthus genome, reflecting the high quality and coverage of our sequencing results. When comparing our data with genomes of four species of Andropogoneae, M. sinensis showed the closest relationship with sugarcane with up to 93 % mapping ratios, followed by sorghum with up to 80 % mapping ratios, indicating a high conservation of orthologs in these three genomes. Furthermore, 306,228 transcripts were successfully annotated against public databases including cell wall related genes and transcript factor families, thus providing many new insights into gene functions. The PacBio Iso-Seq data also helped identify 3,898 alternative splicing events and 2,963 annotated AS isoforms within 10 function categories. CONCLUSIONS: Taken together, the present study provides a rich data set of full-length transcripts that greatly enriches our understanding of M. sinensis transcriptomic resources, thus facilitating further genetic improvement and molecular studies of the Miscanthus species.

Phialocephala fortinii increases aluminum tolerance in Miscanthus sinensis growing in acidic mine soil.

Miscanthus sinensis growing in our study mine site contained a high concentration of Al in the adventitious roots. It has a root endophyte, Phialocephala fortinii, in its adventitious roots at a high frequency. The purpose of this study was to elucidate the effects of P. fortinii on Al tolerance mechanisms of M. sinensis and reveal potential underlying mechanisms. In the absence of P. fortinii, M. sinensis produced chlorogenic, citric, and malic acids that could act to alleviate Al toxicity in acidic mine soil. Up on fungal inoculation, the levels of these compounds were reduced, although the growth of seedlings and Mg concentration in the roots were increased. IAA production by the fungus may contribute to enhanced plant growth whereas an increase of Mg uptake could reduce toxicity of reactive oxygen species under Al stress. These actions of P. fortinii could promote growth and survival of M. sinensis in mine sites.

Chemical Composition of Miscanthus sinensis var. purpurascens Flower Absolute and Its Beneficial Effects on Skin Wound Healing and Melanogenesis-Related Cell Activities.

Miscanthus sinensis var. purpurascens (MSP, flame grass) is found in Korea, Japan, and China, and its biological activities include anti-cancer, detoxifying, vasodilatory, antipyretic, and diuretic effects. However, no study has investigated the effects of MSP on skin-related biological activities. In this study, we explored the effects of the absolute extracted from the MSP flowers (MSPFAb) on skin wound healing- and whitening-related responses in keratinocytes or melanocytes. MSPFAb contained 6 components and induced the proliferation, migration, and syntheses of type I and IV collagens in keratinocytes. MSPFAb also increased the phosphorylations of serine/threonine-specific protein kinase, p38 mitogen-activated protein kinase, and extracellular signal-regulated kinase1/2 in keratinocytes. In addition, treatment with MSPFAb decreased serum-induced melanoma cell proliferation and inhibited tyrosinase activity and melanin contents in α-MSH-stimulated melanoma cells. Taken together, this study indicates MSPFAb may promote wound healing- and whitening-associated activities in dermal cells, and suggests that it has potential use as a wound healing and skin whitening agent.

Lignins Isolated via Catalyst-Free Organosolv Pulping from Miscanthus x giganteus, M. sinensis, M. robustus and M. nagara: A Comparative Study.

As a low-input crop, Miscanthus offers numerous advantages that, in addition to agricultural applications, permits its exploitation for energy, fuel, and material production. Depending on the Miscanthus genotype, season, and harvest time as well as plant component (leaf versus stem), correlations between structure and properties of the corresponding isolated lignins differ. Here, a comparative study is presented between lignins isolated from M. x giganteus, M. sinensis, M. robustus and M. nagara using a catalyst-free organosolv pulping process. The lignins from different plant constituents are also compared regarding their similarities and differences regarding monolignol ratio and important linkages. Results showed that the plant genotype has the weakest influence on monolignol content and interunit linkages. In contrast, structural differences are more significant among lignins of different harvest time and/or season. Analyses were performed using fast and simple methods such as nuclear magnetic resonance (NMR) spectroscopy. Data was assigned to four different linkages (A: ß-O-4 linkage, B: phenylcoumaran, C: resinol, D: ß-unsaturated ester). In conclusion, A content is particularly high in leaf-derived lignins at just under 70% and significantly lower in stem and mixture lignins at around 60% and almost 65%. The second most common linkage pattern is D in all isolated lignins, the proportion of which is also strongly dependent on the crop portion. Both stem and mixture lignins, have a relatively high share of approximately 20% or more (maximum is M. sinensis Sin2 with over 30%). In the leaf-derived lignins, the proportions are significantly lower on average. Stem samples should be chosen if the highest possible lignin content is desired, specifically from the M. x giganteus genotype, which revealed lignin contents up to 27%. Due to the better frost resistance and higher stem stability, M. nagara offers some advantages compared to M. x giganteus. Miscanthus crops are shown to be very attractive lignocellulose feedstock (LCF) for second generation biorefineries and lignin generation in Europe.

Root-associated (rhizosphere and endosphere) microbiomes of the Miscanthus sinensis and their response to the heavy metal contamination.

The plant root-associated microbiomes, including both the rhizosphere and the root endosphere microbial community, are considered as a critical extension of the plant genome. Comparing to the well-studied rhizosphere microbiome, the understanding of the root endophytic microbiome is still in its infancy. Miscanthus sinensis is a pioneering plant that could thrive on metal contaminated lands and holds the potential for phytoremediation applications. Characterizing its root-associated microbiome, especially the root endophytic microbiome, could provide pivotal knowledge for phytoremediation of mine tailings. In the current study, M. sinensis residing in two Pb/Zn tailings and one uncontaminated site were collected. The results demonstrated that the metal contaminant fractions exposed strong impacts on the microbial community structures. Their influences on the microbial community, however, gradually decreases from the bulk soil through the rhizosphere soil and finally to the endosphere, which resulting in distinct root endophytic microbial community structures compared to both the bulk and rhizosphere soil. Diverse members affiliated with the order Rhizobiales was identified as the core microbiome residing in the root of M. sinensis. In addition, enrichment of plant-growth promoting functions within the root endosphere were predicted, suggesting the root endophytes may provide critical services to the host plant. The current study provides new insights into taxonomy and potential functions of the root-associated microbiomes of the pioneer plant, M. sinensis, which may facilitate future phytoremediation practices.

Endophytic Microbiome of Biofuel Plant Miscanthus sinensis (Poaceae) Interacts with Environmental Gradients.

Miscanthus in Taiwan occupies a cline along altitude and adapts to diverse environments, e.g., habitats of high salinity and volcanoes. Rhizospheric and endophytic bacteria may help Miscanthus acclimate to those stresses. The relative contributions of rhizosphere vs. endosphere compartments to the adaptation remain unknown. Here, we used targeted metagenomics to compare the microbial communities in the rhizosphere and endosphere among ecotypes of M. sinensis that dwell habitats under different stresses. Proteobacteria and Actinobacteria predominated in the endosphere. Diverse phyla constituted the rhizosphere microbiome, including a core microbiome found consistently across habitats. In endosphere, the predominance of the bacteria colonizing from the surrounding soil suggests that soil recruitment must have subsequently determined the endophytic microbiome in Miscanthus roots. In endosphere, the bacterial diversity decreased with the altitude, likely corresponding to rising limitation to microorganisms according to the species-energy theory. Specific endophytes were associated with different environmental stresses, e.g., Pseudomonas spp. for alpine and Agrobacterium spp. for coastal habitats. This suggests Miscanthus actively recruits an endosphere microbiome from the rhizosphere it influences.

Identification of candidate reference genes for quantitative RT-PCR in Miscanthus sinensis subjected to various abiotic stresses.

Quantitative real-time PCR (qRT-PCR) has been widely used for studying gene expression at the transcript level. Its accuracy usually relies on the reference genes that are utilized for data normalization. Miscanthus sinensis, a perennial C4 grass with high biomass and strong resistance to adversities, is often utilized as a high value energy crop. However, no reliable reference genes have been investigated for normalizing gene expression for this species. In this study, 12 candidate reference genes were selected to identify their stability under five different abiotic stress treatments (drought, salt, cadmium, chromium and arsenic) by using geNorm, NormFinder, BestKeeper and RefFinder softwares. The results showed that 18S rRNA and Unigene33312 were the best reference genes under drought treatments. Unigene33312 and Unigene33024 were found to be the most stably expressed genes under salt stress and Cd stress. Moreover, Unigene33024 and PP2A were the most suitable reference genes under Cr stress and Unigene33024 and Sb09g019750 were deemed more suitable reference genes under As stress. In total, considering all the samples, Unigene33024 and PP2A were the most stable genes while ACTIN and Unigene26576 were the least stable reference genes for internal control. The expression patterns of two target genes (Cu/Zn SOD and CAT) were used to further verify those selected reference genes under different conditions. The results showed that the most and the least stable reference genes had clearly different expression patterns. This work comprehensively estimated the stability of reference genes in M. sinensis which may give insight to the reference genes selection in other tissues as well as other related varieties. These suggested reference genes would assist in further putative gene expression validation in M. sinensis.

A Two-Step Ferric Chloride and Dilute Alkaline Pretreatment for Enhancing Enzymatic Hydrolysis and Fermentable Sugar Recovery from Miscanthus sinensis.

A two-step process was proposed to enhance enzymatic hydrolysis of Miscanthus sinensis based on a comparative study of acid/alkaline pretreatments. Ferric chloride pretreatment (FP) effectively removed hemicellulose and recovered soluble sugars, but the enzymatic hydrolysis was not efficient. Dilute alkaline pretreatment (ALP) resulted in much better delignification and stronger morphological changes of the sample, making it more accessible to enzymes. While ALP obtained the highest sugar yield during enzymatic hydrolysis, the soluble sugar recovery from the pretreatment stage was still limited. Furthermore, a two-step ferric chloride and dilute alkaline pretreatment (F-ALP) has been successfully developed by effectively recovering soluble sugars in the first FP step and further removing lignin of the FP sample in the second ALP step to improve its enzymatic hydrolysis. As a result, the two-step process yielded the highest total sugar recovery (418.8 mg/g raw stalk) through the whole process.

Genomic index selection provides a pragmatic framework for setting and refining multi-objective breeding targets in Miscanthus.

BACKGROUND: Miscanthus has potential as a biomass crop but the development of varieties that are consistently superior to the natural hybrid M. × giganteus has been challenging, presumably because of strong G × E interactions and poor knowledge of the complex genetic architectures of traits underlying biomass productivity and climatic adaptation. While linkage and association mapping studies are starting to generate long lists of candidate regions and even individual genes, it seems unlikely that this information can be translated into effective marker-assisted selection for the needs of breeding programmes. Genomic selection has emerged as a viable alternative, and prediction accuracies are moderate across a range of phenological and morphometric traits in Miscanthus, though relatively low for biomass yield per se. METHODS: We have previously proposed a combination of index selection and genomic prediction as a way of overcoming the limitations imposed by the inherent complexity of biomass yield. Here we extend this approach and illustrate its potential to achieve multiple breeding targets simultaneously, in the absence of a priori knowledge about their relative economic importance, while also monitoring correlated selection responses for non-target traits. We evaluate two hypothetical scenarios of increasing biomass yield by 20 % within a single round of selection. In the first scenario, this is achieved in combination with delaying flowering by 44 d (roughly 20 %), whereas, in the second, increased yield is targeted jointly with reduced lignin (-5 %) and increased cellulose (+5 %) content, relative to current average levels in the breeding population. KEY RESULTS: In both scenarios, the objectives were achieved efficiently (selection intensities corresponding to keeping the best 20 and 4 % of genotypes, respectively). However, the outcomes were strikingly different in terms of correlated responses, and the relative economic values (i.e. value per unit of change in each trait compared with that for biomass yield) of secondary traits included in selection indices varied considerably. CONCLUSIONS: Although these calculations rely on multiple assumptions, they highlight the need to evaluate breeding objectives and explicitly consider correlated responses in silico, prior to committing extensive resources. The proposed approach is broadly applicable for this purpose and can readily incorporate high-throughput phenotyping data as part of integrated breeding platforms.

Population structure of Miscanthus sacchariflorus reveals two major polyploidization events, tetraploid-mediated unidirectional introgression from diploid M. sinensis, and diversity centred around the Yellow Sea.

BACKGROUND AND AIMS: Miscanthus, a C4 perennial grass native to East Asia, is a promising biomass crop. Miscanthus sacchariflorus has a broad geographic range, is used to produce paper in China and is one of the parents (along with Miscanthus sinensis) of the important biomass species Miscanthus × giganteus. The largest study of M. sacchariflorus population genetics to date is reported here. METHODS: Collections included 764 individuals across East Asia. Samples were genotyped with 34 605 single nucleotide polymorphisms (SNPs) derived from restriction site-associated DNA sequencing (RAD-seq) and ten plastid microsatellites, and were subjected to ploidy analysis by flow cytometry. KEY RESULTS: Six major genetic groups within M. sacchariflorus were identified using SNP data: three diploid groups, comprising Yangtze (M. sacchariflorus ssp. lutarioriparius), N China and Korea/NE China/Russia; and three tetraploid groups, comprising N China/Korea/Russia, S Japan and N Japan. Miscanthus sacchariflorus ssp. lutarioriparius was derived from the N China group, with a substantial bottleneck. Japanese and mainland tetraploids originated from independent polyploidization events. Hybrids between diploid M. sacchariflorus and M. sinensis were identified in Korea, but without introgression into either parent species. In contrast, tetraploid M. sacchariflorus in southern Japan and Korea exhibited substantial hybridization and introgression with local diploid M. sinensis. CONCLUSIONS: Genetic data indicated that the land now under the Yellow Sea was a centre of diversity for M. sacchariflorus during the last glacial maximum, followed by a series of migrations as the climate became warmer and wetter. Overall, M. sacchariflorus has greater genetic diversity than M. sinensis, suggesting that breeding and selection within M. sacchariflorus will be important for the development of improved M. × giganteus. Ornamental M. sacchariflorus genotypes in Europe and North America represent a very narrow portion of the species' genetic diversity, and thus do not well represent the species as a whole.

[Studies on identification of Phragmitis Rhizoma and its counterfeits].

The study aims to explore the main differential characteristics of Phragmites Rhizoma and its counterfeits (rhizomes of Arundo donax, Triarrhena lutarioriparia and Miscanthus sinensis) and provide experimental basis for the reasonable applications of gramineous plants through system research and comparison of plant morphogenesis, character, transverse organization characteristics and powder microscopic characteristics.

Ecological characteristics and in situ genetic associations for yield-component traits of wild Miscanthus from eastern Russia.

Background and aims Miscanthus is a genus of perennial C4 grasses native to East Asia. It includes the emerging ligno-cellulosic biomass crop M. ×giganteus, a hybrid between M. sinensis and M. sacchariflorus. Biomass yield and cold tolerance are of particular interest in Miscanthus, given that this crop is more temperate adapted than its C4 relatives maize, sorghum and sugarcane. Methods A plant exploration was conducted in eastern Russia, at the northern extreme of the native range for Miscanthus, with collections including 174 clonal germplasm accessions (160 M. sacchariflorus and 14 M. sinensis) from 47 sites. Accessions were genotyped by restriction site-associated DNA sequencing (RAD-seq) and plastid microsatellites. Key Results Miscanthus sinensis was found in maritime climates near Vladivostok (43·6°N) and on southern Sakhalin Island (46·6°N). Miscanthus sacchariflorus was found inland at latitudes as high as 49·3°N, where M. sinensis was absent. Most M. sacchariflorus accessions were diploid, but approx. 2 % were tetraploids. Molecular markers revealed little population structure (Jost's D < 0·007 among diploid groups) but high genetic diversity (expected heterozygosity = 0·14) within the collection of Russian M. sacchariflorus. Genome-wide association (GWA) analysis for traits measured at the collection sites revealed three M. sacchariflorus single nucleotide polymorphisms (SNPs) significantly associated with the number of stems per unit area, one with height and one with basal stem diameter; three were near or within previously described sorghum quantitative trait loci for related traits. Conclusions This new Miscanthus germplasm collection from eastern Russia will be useful for breeding Miscanthus and sugarcane cultivars with improved adaptation to cold. Moreover, a strategy is proposed to facilitate the rapid utilization of new germplasm collections: by implementing low-cost SNP genotyping to conduct GWA studies of phenotypic data obtained at collection sites, plant breeders can be provided with actionable information on which accessions have desirable traits and alleles.

Contrasting allelic distribution of CO/Hd1 homologues in Miscanthus sinensis from the East Asian mainland and the Japanese archipelago.

The genus Miscanthus is a perennial C4 grass native to eastern Asia and is a promising candidate bioenergy crop for cool temperate areas. Flowering time is a crucial factor governing regional and seasonal adaptation; in addition, it is also a key target trait for extending the vegetative phase to improve biomass potential. Homologues of CONSTANS (CO)/Heading date 1(Hd1) were cloned from Miscanthus sinensis and named MsiHd1. Sequences of MsiHd1 homologues were compared among 24 wild M. sinensis accessions from Japan, 14 from China, and three from South Korea. Two to five MsiHd1 alleles in each accession were identified, suggesting that MsiHd1 consists of at least three loci in the Miscanthus genome. Verifying the open reading frame in MsiHd1, they were classified as putative functional alleles without mutations or non-functional alleles caused by indels. The Neighbor-Joining tree indicated that one of the multiple MsiHd1 loci is a pseudogene locus without any functional alleles. The pseudogene locus was named MsiHd1b, and the other loci were considered to be part of the MsiHd1a multi-locus family. Interestingly, in most Japanese accessions 50% or more of the MsiHd1a alleles were non-functional, whereas accessions from the East Asian mainland harboured only functional alleles. Five novel miniature inverted transposable elements (MITEs) (MsiMITE1-MsiMITE5) were observed in MsiHd1a/b. MsiMITE1, detected in exon 1 of MsiHd1a, was only observed in Japanese accessions and its revertant alleles derived from retransposition were predominantly in Chinese accessions. These differences in MsiHd1a show that the dependency on functional MsiHd1a alleles is different between accessions from the East Asian mainland and Japan.

Can the exceptional chilling tolerance of C4 photosynthesis found in Miscanthus × giganteus be exceeded? Screening of a novel Miscanthus Japanese germplasm collection.

BACKGROUND AND AIMS: A clone of the hybrid perennial C4 grass Miscanthus × giganteus (Mxg) is known for achieving exceptionally high rates of leaf CO2 uptake during chilling. This is a requisite of success in the early spring, as is the ability of the leaves to survive occasional frosts. The aim of this study was to search for genotypes with greater potential than Mxg for photosynthesis and frost survival under these conditions. METHODS: A total of 864 accessions representing 164 local populations of M. sacchariflorus (Msa), M. sinensis (Msi) and M. tinctorius (Mti) collected across Japan were studied. Accessions whose leaves survived a natural late frost in the field were screened for high maximum photosystem II efficiency (Fv/Fm) following chilling weather, as an indicator of their capacity for light-limited photosynthesis. Those showing the highest Fv/Fm were transferred to a high-light-controlled environment and maintained at chilling temperatures, where they were further screened for their capacities for high-light-limited and light-saturated leaf uptake of CO2 (ΦCO2,max and Asat, respectively). KEY RESULTS: For the first time, relatives of Mxg with significantly superior capacities for photosynthesis at chilling temperatures were identified. Msa accession '73/2' developed leaves in the spring that survived night-time frost, and during growth under chilling maintained a statistically significant 79 % higher ΦCO2,max, as a measure of light-limited photosynthesis, and a 70 % higher Asat, as a measure of light-saturated photosynthesis. A second Msa accession, '73/3' also showed significantly higher rates of leaf uptake of CO2. CONCLUSIONS: As remarkable as Mxg has proved in its chilling tolerance of C4 photosynthesis, this study shows that there is still value and potential in searching for yet more superior tolerance. Msa accession '73/2' shows rates of light-limited and light-saturated photosynthesis at chilling temperatures that are comparable with those of the most cold-tolerant C3 species. This adds further proof to the thesis that C4 photosynthesis is not inherently limited to warm climates.

Potential use of Pseudomonas koreensis AGB-1 in association with Miscanthus sinensis to remediate heavy metal(loid)-contaminated mining site soil.

Endophytic bacteria have the potential to promote plant growth and heavy metal(loid) (HM) removal from contaminated soil. Pseudomonas koreensis AGB-1, isolated from roots of Miscanthus sinensis growing in mine-tailing soil, exhibited high tolerance to HMs and plant growth promoting traits. Transmission electron microscope (TEM) analysis revealed that AGB-1 sequestered HMs extracellularly and their accumulation was visible as dark metal complexes on bacterial surfaces and outside of the cells. DNA sequencing of HM resistance marker genes indicated high homology to the appropriate regions of the arsB, ACR3(1), aoxB, and bmtA determinants. Inoculating mining site soil with AGB-1 increased M. sinensis biomass by 54%, chlorophyll by 27%, and protein content by 28%. High superoxide dismutase and catalase activities, and the lower malondialdehyde content of plants growing in AGB-1-inoculated soil indicate reduced oxidative stress. Metal(loid) concentrations in roots and shoots of plants grown in inoculated soil were higher than those of the controls in pot trials with mine tailing soil. Results suggest that AGB-1 can be used in association with M. sinensis to promote phytostabilization and remediation of HM-contaminated sites.

Genome-wide association studies and prediction of 17 traits related to phenology, biomass and cell wall composition in the energy grass Miscanthus sinensis.

• Increasing demands for food and energy require a step change in the effectiveness, speed and flexibility of crop breeding. Therefore, the aim of this study was to assess the potential of genome-wide association studies (GWASs) and genomic selection (i.e. phenotype prediction from a genome-wide set of markers) to guide fundamental plant science and to accelerate breeding in the energy grass Miscanthus. • We generated over 100,000 single-nucleotide variants (SNVs) by sequencing restriction site-associated DNA (RAD) tags in 138 Micanthus sinensis genotypes, and related SNVs to phenotypic data for 17 traits measured in a field trial. • Confounding by population structure and relatedness was severe in naïve GWAS analyses, but mixed-linear models robustly controlled for these effects and allowed us to detect multiple associations that reached genome-wide significance. Genome-wide prediction accuracies tended to be moderate to high (average of 0.57), but varied dramatically across traits. As expected, predictive abilities increased linearly with the size of the mapping population, but reached a plateau when the number of markers used for prediction exceeded 10,000-20,000, and tended to decline, but remain significant, when cross-validations were performed across subpopulations. • Our results suggest that the immediate implementation of genomic selection in Miscanthus breeding programs may be feasible.

Variation in chilling tolerance for photosynthesis and leaf extension growth among genotypes related to the C4 grass Miscanthus ×giganteus.

The goal of this study was to identify cold-tolerant genotypes within two species of Miscanthus related to the exceptionally chilling-tolerant C4 biomass crop accession: M. ×giganteus 'Illinois' (Mxg) as well as in other Mxg genotypes. The ratio of leaf elongation at 10 °C/5 °C to that at 25 °C/25 °C was used to identify initially the 13 most promising Miscanthus genotypes out of 51 studied. Net leaf CO2 uptake (A sat) and the maximum operating efficiency of photosystem II (ФPSII) were measured in warm conditions (25 °C/20 °C), and then during and following a chilling treatment of 10 °C/5 °C for 11 d. Accessions of M. sacchariflorus (Msa) showed the smallest decline in leaf elongation on transfer to chilling conditions and did not differ significantly from Mxg, indicating greater chilling tolerance than diploid M. sinensis (Msi). Msa also showed the smallest reductions in A sat and ФPSII, and greater chilling-tolerant photosynthesis than Msi, and three other forms of Mxg, including new triploid accessions and a hexaploid Mxg 'Illinois'. Tetraploid Msa 'PF30153' collected in Gifu Prefecture in Honshu, Japan did not differ significantly from Mxg 'Illinois' in leaf elongation and photosynthesis at low temperature, but was significantly superior to all other forms of Mxg tested. The results suggested that the exceptional chilling tolerance of Mxg 'Illinois' cannot be explained simply by the hybrid vigour of this intraspecific allotriploid. Selection of chilling-tolerant accessions from both of Mxg's parental species, Msi and Msa, would be advisable for breeding new highly chilling-tolerant Mxg genotypes.