Photosynthetic variation and detoxification strategies based on cadmium uptake, non-protein thiols, and secondary metabolites in Miscanthus sacchariflorus under cadmium exposure.

Miscanthus sacchariflorus is previously demonstrated to be a potential candidate for remediation of cadmium (Cd) pollution. To explore its resistance strategy to Cd, a hydroponic experiment was conducted to determine the variations of photosynthetic activity in leaves and physiological response in roots of this plant. Results showed that the root of M. sacchariflorus was the primary location for Cd accumulation. The bioconcentration factor in the roots and rhizomes was >1, and the translocation factor from underground to aboveground was <1. Throughout the experimental period, treatment with 0.06 mM Cd2+ did not significantly alter the contents of chlorophyll a, chlorophyll b, or carotenoid. By contrast, treatment with 0.15 and 0.30 mM Cd2+ decreased the contents of chlorophyll a, chlorophyll b, and carotenoid; caused the deformation of the chlorophyll fluorescence transient curve; reduced the photochemical efficiency of photosystem II; and increased the contents of non-protein thiols, total flavone, and total phenol. These results indicate that M. sacchariflorus has good adaptability to 0.06 mM Cd2+. Moreover, the accumulation of the non-protein thiols, total flavone, and total phenol in roots may promote the chelation of Cd2+, thus alleviating Cd toxicity. This study provides theoretical support for using M. sacchariflorus to remediate Cd-polluted wetlands.

Effects of hydrogen sulfide on the growth and physiological characteristics of Miscanthus sacchariflorus seedlings under cadmium stress.

As a gas signal molecule, hydrogen sulfide (H2S) can participate in many physiological and biochemical processes such as seed germination and photosynthesis regulation. In order to explore the regulatory effect of H2S on the growth of Miscanthus sacchariflorus under Cd stress and to provide sufficient theoretical basis for the complex action of H2S and energy plants to remediate soil pollution. In this experiment, the effects of different concentrations of H2S (10, 25, 50, 100, 300, 400, 500 µmol·L-1 (µM)) pretreatment on the growth index, lipid peroxidation degree, chlorophyll (Chl) content, osmoregulation substance content, antioxidant enzyme activity and non-enzymatic antioxidant content of M. sacchariflorus under Cd stress (50 µM) were studied. The results showed that under Cd stress, the reactive oxygen species (ROS) content in the body of M. sacchariflorus was unbalanced, and the growth were severely inhibited, the activities of antioxidant enzymes, such as catalase (CAT) and superoxide dismutase (SOD) significantly decreased, and the content of osmoregulation substance, ascorbic acid (AsA) and glutathione (GSH) significantly increased. With the increase of H2S concentration, its effect on resisting Cd stress can be shown as "low concentration promotes, high concentration inhibits". When the concentration of H2S ≤ 300 µM, although there was no significant difference in Cd content compared with Cd treatment alone, it can regulate the activities of peroxidase (POD), SOD, glutathione reductase (GR) and monodehydroascorbate reductase (MDHAR), increase the content of osmoregulation substances, oxidized glutathione (GSSG), and the transformation rate of AsA and dehydroascorbic acid (DHA) to reduce the oxidative damage and improve the growth and photosynthetic indicators of plants; when the concentration of H2S ≥ 400 µM, Cd content in the ground and root decreased significantly, but the transport factor increased significantly, the growth status of M. sacchariflorus were more severely inhibited by the combined stress of H2S and Cd. In this experiment, it was found that the concentration of H2S pretreatment ≤ 300 µM could regulate the growth of M. sacchariflorus under Cd stress to normal level, and when the treatment concentration was 50 µM, the effect was the best. It will provide a new idea for the treatment of contaminated soil by energy plants.

Reference gene selection for quantitative RT-PCR in Miscanthus sacchariflorus under abiotic stress conditions.

BACKGROUND: Reference genes are necessary for quantitative real-time PCR (qRT-PCR) analysis and their stability can directly influence the accuracy of gene expression result. Miscanthus sacchariflorus, a perennial C4 grass that serves as promising biofuel plant for temperate climates, has not been explored for the identification of stable reference genes yet. MATERIALS AND METHODS: Nine potential reference genes (ACT, EF1a, FBOX, GAPDH, PP2A, SAND, TIP41, TUB and UBC) of M. sacchariflorus under different abiotic (salinity, drought and cadmium) stresses, as well as in two tissues (roots and leaves) were evaluated. The expression stability of these genes were analyzed by four commonly used software programs (geNorm, NormFinder, BestKeeper, ΔCt method and RefFinder). RESULTS: Our results found that FBOX and SAND are the most stable genes among all tested samples. FBOX and EF1a are suitable for gene expression normalization of cadmium-treated samples and salinity-treated leaves. FBOX and PP2A are appropriate reference genes for salt-stressed roots and PEG-treated leaves. The traditional reference gene ACT and GAPDH exhibited the most variable pattern, which would not be recommended for qRT-PCR analysis under different abiotic stresses. Furthermore, the expression levels of PIP2, NHX1 and MT2a under drought, salt and cadmium treatment were detected with above reference genes. CONCLUSIONS: This work identified the appropriate reference genes for qRT-PCR in M. sacchariflorus and FBOX was recommended to be effective internal control for gene expression normalization in M. sacchariflorus in response to different abiotic stresses.

Comparative transcriptome analysis and identification of candidate adaptive evolution genes of Miscanthus lutarioriparius and Miscanthus sacchariflorus.

Miscanthus species are perennial C4 grasses that are considered promising energy crops because of their high biomass yields, excellent adaptability and low management costs. Miscanthus lutarioriparius and Miscanthus sacchariflorus are closely related subspecies that are distributed in different habitats. However, there are only a few reports on the mechanisms by which Miscanthus adapts to different environments. Here, comparative transcriptomic and morphological analyses were used to study the evolutionary adaptation of M. lutarioriparius and M. sacchariflorus to different habitats. In total, among 7586 identified orthologs, 2060 orthologs involved in phenylpropanoid biosynthesis and plant hormones were differentially expressed between the two species. Through an analysis of the Ka/Ks ratios of the orthologs, we estimated that the divergence time between the two species was approximately 4.37 Mya. In addition, 37 candidate positively selected orthologs (PSGs) that played important roles in the adaptation of these species to different habitats were identified. Then, the expression levels of 20 PSGs in response to flooding and drought stress were analyzed, and the analysis revealed significant changes in their expression levels. These results facilitate our understanding of the evolutionary adaptation to habitats and the speciation of M. lutarioriparius and M. sacchariflorus. We hypothesise that lignin synthesis genes are the main cause of the morphological differences between the two species. In summary, the plant nonspecific phospholipase C gene family and the receptor-like protein kinase gene family played important roles in the evolution of these two species. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01030-1.

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.

Comparative study on effects of four energy plants growth on chemical fractions of heavy metals and activity of soil enzymes in copper mine tailings.

Four gramineous energy plants, Miscanthus sacchariflorus, M. floridulus, Phragmites australis, and Arundo donax were grown on copper tailings in the field for four years. Their phytoremediation potential was examined in terms of their effects on the fractions of heavy metals and soil enzyme activities. Results showed that plantation of these four gramineous plants has improved the proportion of organic material (OM)-binding fraction of heavy metals in copper tailings as a whole, and reduced the proportion of exchangeable and residual fractions. In particular, M. sacchariflorus growth improved significantly the proportion of the OM-binding fractions of Cu (1.73 times), Cd (1.71 times), Zn (1.18 times), and Pb (3.14 times) (P < 0.05) and reduced markedly the residual fractions of Cu (64.45%), Cd (82.38%), Zn (61.43%), and Pb (73.41%) (P < 0.05). Except for A. donax, the growth of other three energy plants improved the activity of phosphatase, urease and dehydrogenase in copper tailings to some extent. In particular, the activity of soil phosphatase and urease in planted tailings differed significantly from that of control (P < 0.05). The effect of M. sacchariflorus growth on soil enzyme was the highest, followed by P. australis, M. floridulus, and A. donax. The content of each heavy metal fraction in soil was correlated with soil enzyme activities, especially the content of OM-binding fraction, which correlated significantly with the activities of phosphatase, urease and dehydrogenase in soil. According to the effects of four gramineous plants growth on activity of soil enzymes and fractions of heavy metals, M. sacchariflorus had the optimal effects for phytoremediation. Therefore, M. sacchariflorus was a candidate plant with great potential for the revegetation of heavy metal tailings.

Exogenous malic acid alleviates cadmium toxicity in Miscanthus sacchariflorus through enhancing photosynthetic capacity and restraining ROS accumulation.

Malic acid (MA) plays an important role in the regulation of plant growth, stomatal aperture, nutrition elements homeostasis and toxic metals tolerance. However, little is known about the effects of exogenous MA on physiological and biochemical responses to toxic metals in plants. To measure the alleviation roles of exogenous MA against cadmium (Cd), we determined the effects of MA on plant growth, net photosynthetic rate (Pn), reactive oxygen species (ROS) accumulation and the activities of anti-oxidant enzymes in the leaves of Miscanthus sacchariflorus (M. sacchariflorus) under Cd stress. The Cd exposure alone significantly inhibited plant growth and Pn, but increased the accumulation of ROS even though the anti-oxidant enzymes were markedly activated in the leaves of M. sacchariflorus. Treatment with MA significantly enhanced plant growth and decreased Cd accumulation accompanied by increasing Pn under Cd stress as compared to Cd stress alone, especially when treatment with high concentration of MA (200µM) was used. In addition, Cd and MA indicated synergistic effects by further increasing the activities and genes expression of partial anti-oxidant enzymes, thus resulting in higher glutathione accumulation and reduction of ROS production. The results showed that application of MA alleviated Cd-induced phytotoxicity and oxidant damage through the regulation of both enzymatic and non-enzymatic anti-oxidants under Cd stress in M. sacchariflorus.

Real-time kinetics of cadmium transport and transcriptomic analysis in low cadmium accumulator Miscanthus sacchariflorus.

MAIN CONCLUSION: The molecular mechanism of low Cd influxes and accumulation in Miscanthus sacchariflorus is revealed by RNA sequencing technique. Soil cadmium (Cd) pollution has posed a serious threat to our soil quality and food security as well as to human health. Some wild plants exhibit high tolerance to heavy metals stress. However, mechanisms of Cd tolerance of wild plants remain to be fully clarified. In this study, we found that two Miscanthus species, Miscanthus (M.) sacchariflorus and M. floridulus, showed different Cd-tolerant mechanisms. M. sacchariflorus accumulated less Cd in both root and leaf by limiting Cd uptake from root and showed superior Cd tolerance, while M. floridulus not only absorbs more Cd from root but also transports more Cd to shoot. To investigate the molecular mechanism of different Cd uptake patterns in the two Miscanthus species, we analyzed the transcriptome of M. sacchariflorus and identified transcriptional changes in response to Cd in roots by high-throughput RNA-sequencing technology. A total of 92,985 unigenes were obtained from M. sacchariflorus root cDNA samples. Based on the assembled de novo transcriptome, 681 DEGs which included 345 upregulated and 336 downregulated genes were detected between two libraries of untreated and Cd-treated roots. Gene ontology (GO) and pathway enrichment analysis revealed that upregulated DEGs under Cd stress are predominately involved in metabolic pathway, starch and sucrose and biosynthesis of secondary metabolites and metal ion transporters. Quantitative RT-PCR was employed to compare the expression levels of some metal transport genes in roots of two Miscanthus species, and the genes involved in Cd uptake from root and transfer from root to shoot were extremely different. The results not only enrich genomic resource but also help to better understand the molecular mechanisms of Cd accumulation and tolerance in wild plants.

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.

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.

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.

Salt tolerance evaluation and mini-core collection development in Miscanthus sacchariflorus and M. lutarioriparius.

Marginal lands, such as those with saline soils, have potential as alternative resources for cultivating dedicated biomass crops used in the production of renewable energy and chemicals. Optimum utilization of marginal lands can not only alleviate the competition for arable land use with primary food crops, but also contribute to bioenergy products and soil improvement. Miscanthus sacchariflorus and M. lutarioriparius are prominent perennial plants suitable for sustainable bioenergy production in saline soils. However, their responses to salt stress remain largely unexplored. In this study, we utilized 318 genotypes of M. sacchariflorus and M. lutarioriparius to assess their salt tolerance levels under 150 mM NaCl using 14 traits, and subsequently established a mini-core elite collection for salt tolerance. Our results revealed substantial variation in salt tolerance among the evaluated genotypes. Salt-tolerant genotypes exhibited significantly lower Na+ content, and K+ content was positively correlated with Na+ content. Interestingly, a few genotypes with higher Na+ levels in shoots showed improved shoot growth characteristics. This observation suggests that M. sacchariflorus and M. lutarioriparius adapt to salt stress by regulating ion homeostasis, primarily through enhanced K+ uptake, shoot Na+ exclusion, and Na+ sequestration in shoot vacuoles. To evaluate salt tolerance comprehensively, we developed an assessment value (D value) based on the membership function values of the 14 traits. We identified three highly salt-tolerant, 50 salt-tolerant, 127 moderately salt-tolerant, 117 salt-sensitive, and 21 highly salt-sensitive genotypes at the seedling stage by employing the D value. A mathematical evaluation model for salt tolerance was established for M. sacchariflorus and M. lutarioriparius at the seedling stage. Notably, the mini-core collection containing 64 genotypes developed using the Core Hunter algorithm effectively represented the overall variability of the entire collection. This mini-core collection serves as a valuable gene pool for future in-depth investigations of salt tolerance mechanisms in Miscanthus.

Transcriptomic Characterization of Miscanthus sacchariflorus × M. lutarioriparius and Its Implications for Energy Crop Development in the Semiarid Mine Area.

Miscanthus interspecific hybrids have been proved to have better adaptability in marginal lands than their parents. Miscanthus sacchariflorus and Miscanthus lutarioriparius were used as the parents to develop hybrids. We performed the transcriptome for 110 F1 hybrids of Miscanthus sacchariflorus × Miscanthus lutarioriparius and their parents that had been established on the Loess Plateau mine area, to estimate the population's genetic expression variation, and illuminate the adaptive mechanism of the F1 population. The result speculated that the F1 population has mainly inherited the stress response metabolic pathway of its female parent (M. sacchariflorus), which may be responsible for its higher environmental adaptability and biomass yield compared with male parents. Based on PopART, we assembled a leaf reference transcriptome for M. sacchariflorus (LRTMS) and obtained 8116 high-quality transcripts. When we analyze the differential expression of genes between F1 population and parent, 39 and 56 differentially expressed genes were screened out in the female parent and male parent, respectively. The enrichment analysis showed that pathways of carbohydrate metabolism, lipid metabolism, biosynthesis of secondary metabolites and circadian rhythm-plant played a key role in resisting the harsh environment. The carbohydrate metabolism and lipid metabolism were also significantly enriched, and the synthesis of these substances facilitated the yield. The results provided an insight into breeding Miscanthus hybrids more suited to the harsh environment of the Loess Plateau.

Colonization and phytoremediation potential for Miscanthus sacchariflorus in copper tailings.

A pot experiment was conducted to explore the effects of copper (Cu) tailings with various proportions in the substrate on seed germination and morphological traits of the plant. Concurrently, to identify the adaptive and tolerance strategies of the plant to Cu tailings, the uptake and accumulation of the plant to heavy metals, variations in soil enzymatic activities, and metal speciation in the blank, rhizospheric, and non-rhizospheric soils were estimated. Cu tailings at 25% proportion in the substrate exerted no significant negative effects on seed germination and seedling growth. However, Cu tailings at higher proportions (≧50%) inhibited seed germination and disturbed the plant physiological metabolism and growth. More biomass allocated to the plant roots could contribute to more heavy metals being immobilized, arresting their translocation from roots to shoots. This was accepted as a crucial defense mechanism for the plant against heavy metal co-contamination. The plant can improve the biological properties of Cu tailings in terms of enhanced invertase and phosphatase activities. And in turn, this can effectively alleviate heavy metal phytotoxicity. Simultaneously, it markedly decreased exchangeable Cu and Zn content in the rhizosphere in 25% Cu tailings treatments. In 50% Cu tailings treatment, no differences were observed in Zn speciation between rhizosphere and non-rhizosphere soils. In 75% Cu tailings, compared to the non-rhizosphere, an obvious reduction in exchangeable Cu in rhizosphere occurred, while an opposite tendency was demonstrated in carbonate-bound Zn. The plant could successfully colonize in Cu tailings, and represent a phytoremediation potential in Cu tailings.

Development of energy plants from hybrids between Miscanthus sacchariflorus and M. lutarioriparius grown on reclaimed mine land in the Loess Plateau of China.

Miscanthus, a promising bioenergy plant, has a high biomass yield with high cellulose content suitable for biofuel production. However, harsh climatic and poor soil conditions, such as barren lands or abandoned mines, pose a challenge to the survival and yield of Miscanthus feedstock on the marginal land. The selection from the interspecific hybrids of Miscanthus might combine high survival rates and high yield, which benefits energy crop development in multi-stressful environments. A total of 113 F1 hybrids between Miscanthus sacchariflorus and M. lutarioriparius together with the parents were planted and evaluated for multiple morphological and physiological traits on the mine land of the Loess Plateau of China. The majority of hybrids had higher establishment rates than M. sacchariflorus while M. lutarioriparius failed to survive for the first winter. Nearly all hybrid genotypes outperformed M. lutarioriparius for yield-related traits including plant height, tiller number, tiller diameter, and leaf area. The average biomass of the hybrids was 20 times higher than that of surviving parent, M. sacchariflorus. Furthermore, the photosynthetic rates and water use efficiency of the hybrids were both significantly higher than those of the parents, which might be partly responsible for their higher yield. A total of 29 hybrids with outstanding traits related to yield and stress tolerance were identified as candidates. The study investigated for the first time the hybrids between local individuals of M. sacchariflorus and high-biomass M. lutarioriparius, suggesting that this could be an effective approach for high-yield energy crop development on vast of marginal lands.

Transformation and gene editing in the bioenergy grass Miscanthus.

BACKGROUND: Miscanthus, a C4 member of Poaceae, is a promising perennial crop for bioenergy, renewable bioproducts, and carbon sequestration. Species of interest include nothospecies M. x giganteus and its parental species M. sacchariflorus and M. sinensis. Use of biotechnology-based procedures to genetically improve Miscanthus, to date, have only included plant transformation procedures for introduction of exogenous genes into the host genome at random, non-targeted sites. RESULTS: We developed gene editing procedures for Miscanthus using CRISPR/Cas9 that enabled the mutation of a specific (targeted) endogenous gene to knock out its function. Classified as paleo-allopolyploids (duplicated ancient Sorghum-like DNA plus chromosome fusion event), design of guide RNAs (gRNAs) for Miscanthus needed to target both homeologs and their alleles to account for functional redundancy. Prior research in Zea mays demonstrated that editing the lemon white1 (lw1) gene, involved in chlorophyll and carotenoid biosynthesis, via CRISPR/Cas9 yielded pale green/yellow, striped or white leaf phenotypes making lw1 a promising target for visual confirmation of editing in other species. Using sequence information from both Miscanthus and sorghum, orthologs of maize lw1 were identified; a multi-step screening approach was used to select three gRNAs that could target homeologs of lw1. Embryogenic calli of M. sacchariflorus, M. sinensis and M. x giganteus were transformed via particle bombardment (biolistics) or Agrobacterium tumefaciens introducing the Cas9 gene and three gRNAs to edit lw1. Leaves on edited Miscanthus plants displayed the same phenotypes noted in maize. Sanger sequencing confirmed editing; deletions in lw1 ranged from 1 to 26 bp in length, and one deletion (433 bp) encompassed two target sites. Confocal microscopy verified lack of autofluorescence (chlorophyll) in edited leaves/sectors. CONCLUSIONS: We developed procedures for gene editing via CRISPR/Cas9 in Miscanthus and, to the best of our knowledge, are the first to do so. This included five genotypes representing three Miscanthus species. Designed gRNAs targeted all copies of lw1 (homeologous copies and their alleles); results also confirmed lw1 made a good editing target in species other than Z. mays. The ability to target specific loci to enable endogenous gene editing presents a new avenue for genetic improvement of this important biomass crop.

Screening of Allelochemicals in Miscanthus sacchariflorus Extracts and Assessment of Their Effects on Germination and Seedling Growth of Common Weeds.

There is increasing interest in the application of bioherbicides because they are less destructive to the global ecosystem than synthetic herbicides. Research has focused on reducing the dependence upon synthetic herbicides by substituting them with environmentally and economically sustainable bioproducts. Allelopathic phytochemicals may be an efficient method for controlling weeds, benefitting both the environment and human health. This study addressed the allelopathic potential of Miscanthus sacchariflorus (MS) extracts on the germination, plant growth, biomass, and biochemical parameters (electrolyte leakage, photosynthetic pigments, and antioxidant enzyme activities) of weeds using laboratory and field experiments. Liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) showed the presence of 22 phenolic compounds, including Orientin, Luteolin, Veratric acid, Chlorogenic acid, Protocatechuic acid, p-Coumaric acid, and Ferulic acid. Leaf extracts of M. sacchariflorus either completely suppressed or partially reduced seed germination and affected the development of weed seedlings (root and shoot length), in a dose-dependent manner. Aqueous extracts of M. sacchariflorus reduced the fresh weight and dry weight, affected the photosynthetic pigment content (chlorophylls, carotenoids), influenced the electrolyte ion leakage, and stimulated the activity of antioxidant enzymes in a species-specific manner. Pearson's correlation analysis showed that the phenolic compound composition of M. sacchariflorus correlated with the variables tested, indicating that the phytochemicals present in the plant extracts of M. sacchariflorus are a potential source of bio-herbicides.

Fractionation and characterization of lignins from Miscanthus via organosolv and soda pulping for biorefinery applications.

The structural complexity of lignins necessitates characterization and isolation methodologies for assessing their appropriateness for thermo-chemical systems and material applications. Lignins prepared via two pulping methods (organosolv and soda) were comprehensively investigated by analyzing the properties, including lignin purity, yield, and thermal and chemical properties. The extracted organosolv lignin has high purity (93.13-98.12%), however, the purity of soda lignin was relatively low (87.58-89.61%). Organosolv lignin produced the highest heating value of 26.79-26.95 MJ kg-1, with a fixed carbon content of 39.47-41.06 wt%, high purity, and low ash content, making it suitable for biofuel applications. The content of total phenolic OH groups was higher for the organosolv lignins; however, for the phenolic OH groups, the 4-vinylphenol content was significantly higher in the soda lignins, and increased with increasing NaOH concentration. Overall, the thermal and chemical properties related to the lignin structure changed with the fractionation method and solvent concentration, which in turn influences the design of lignin valorization strategies for prospective depolymerization and material applications.

Silver grass-derived activated carbon with coexisting micro-, meso- and macropores as excellent bioanodes for microbial colonization and power generation in sustainable microbial fuel cells.

In this study, highly biocompatible three-dimensional hierarchically porous activated carbon from the low-cost silver grass (Miscanthus sacchariflorus) has been fabricated through a facile carbonization approach and tested it as bioanode in microbial fuel cell (MFC) using Escherichia coli as biocatalyst. This silver grass-derived activated carbon (SGAC) exhibited an unprecedented specific surface area of 3027 m2 g-1 with the coexistence of several micro-, meso-, and macropores. The synergistic effect from pore structure (macropores - hosting E. coli to form biofilm and facilitates internal mass transfer; mesopores - favors fast electron transfer; and micropores - promotes nutrient transport to the biofilm) with very high surface area facilitates excellent extracellular electron transfer (EET) between the anode and biofilm which resulted in higher power output of 963 mW cm-2. Based on superior biocompatibility, low cost, environment-friendliness, and facile fabrication, the proposed SGAC bioanode could have a great potential for high-performance and cost-effective sustainable MFCs.

Development and economic analysis of bioethanol production facilities using lignocellulosic biomass.

An integrated process for bioethanol production from Miscanthus sacchariflorus was used to construct a bench-scale plant constructed and an economic analysis was carried out to investigate the feasibility of its application to a commercial plant. The bench-scale plant was operated for 1 month and an economic analysis and sensitivity analysis was performed on the data acquired. In this study, 100,000 kL of bioethanol could be produced annually from 606,061 tons of M. sacchariflorus and the production cost was calculated to be US$1.76/L. However, the by-products of this process such as xylose molasses and lignin can be sold or used as a heat source, which can decrease the ethanol production costs. Therefore, the final ethanol production cost was calculated to be US$1.31/L, and is considerably influenced by the enzyme cost. The results and data obtained should contribute to the development of a commercial-scale lignocellulosic bioethanol plant.