Rhizophagus irregularis and biochar can synergistically improve the physiological characteristics of saline-alkali resistance of switchgrass.

Inoculation of arbuscular mycorrhizal fungi (AMF) or biochar (BC) application can improve photosynthesis and promote plant growth under saline-alkali stress. However, little is known about the effects of the two combined on growth and physiological characteristics of switchgrass under saline-alkali stress. This study examined the effects of four treatments: (1) no AMF inoculation and no biochar addition (control), (2) biochar (BC) alone, (3) AMF (Rhizophagus irregularis, Ri) alone, and (4) the combination of both (BC+Ri) on the plant biomass, antioxidant enzymes, chlorophyll, and photosynthetic parameters of switchgrass under saline-alkali stress. The results showed that the above-ground, belowground and total biomass of switchgrass in the BC+Ri treatment group was significantly higher (+136.7%, 120.2% and 132.4%, respectively) than in other treatments compared with Control. BC+Ri treatment significantly increased plant leaves' relative chlorophyll content, antioxidant enzyme activity, and photosynthesis parameters. It is worth noting that the transpiration rate, stomatal conductance, net photosynthetic rate, PSII efficiency and other photosynthetic-related indexes of the BC+Ri treatment group were the highest (38% to 54% higher than other treatments). The fitting results of light response and CO2 response curves showed that the light saturation point, light compensation point, maximum carboxylation rate and maximum electron transfer rate of switchgrass in the Ri+BC treatment group were the highest. In conclusion, biochar combined with Ri has potential beneficial effects on promoting switchgrass growth under saline-alkali stress and improving the activity of antioxidant enzymes and photosynthetic characteristics of plants.

The effect of AMF combined with biochar on plant growth and soil quality under saline-alkali stress: Insights from microbial community analysis.

Arbuscular mycorrhizal fungi (AMF) and biochar application individually can enhance plant tolerance to saline-alkali stress and promote plant growth efficiency. However, little is known about the potential synergistic effects of their combination on improving plant growth and soil quality under saline-alkali stress. This experiment adopted the potted method to explore the effects of four treatments on switchgrass growth and soil quality: biochar (BC), Rhizophagus irregularis (Ri), biochar + Ri (BR) and a control without biochar or Ri (CK). Compared to the CK treatment, the switchgrass biomass increased by 92.4 %, 148.6 %, and 177.3 % in the BC, Ri, and BR treatment groups, respectively. Similarly, the rhizosphere soil quality index increased by 29.33 %, 22.7 %, and 49.1 % in the respective treatment groups. The BR treatment significantly altered the rhizosphere soil microbial composition and diversity. Notably, compared to the other treatments, the archaeal α-diversity in the BR group showed a significant decrease. BR treatment significantly increased the relative abundance of bacteria, fungi and archaea at the genus level (e.g., Bacillus, Trichome and candidatus_methanopenens). Network analysis showed that the complexity and closeness of interactions between different microbial taxa were stronger in the BC, Ri and BR treatments than in the CK treatment, with BR being the more prominent. In summary, biochar combined with Ri has a better effect on promoting the growth of switchgrass under saline-alkali stress, improving the quality of saline-alkali soil, and increasing soil microbial diversity. This study provides a new approach for the efficient development and utilization of saline-alkali land.

Botanical and chemical fungicides in the treatment of commercial seeds of Brachiaria brizantha and Panicum maximum.

Brazil is the largest producer, consumer, and exporter of forage seeds in the world. However, the high incidence of pathogens threatens the seed production system, resulting in decreases in production and quality. Currently, the emphasis has been placed on the use of alternative products as a way of maintaining productivity and environmental sustainability. Thus, the objective of this study was to evaluate the mycoflora associated with the commercial seeds of Brachiaria brizantha and Panicum maximum treated with chemical and alternative fungicide. To test with chemical and alternative treatments, seeds were subjected to fluazinam + thiophanate-methyl, fludioxonil + metalaxyl-M, noni essential oil, rosemary pepper essential oil, sulfuric acid. There was also an untreated control. The most efficient products in the control of incident fungi were, in decreasing order, fluazinam + thiophanate-methyl, fludioxonil + metalaxyl-M, noni essential oil, rosemary pepper essential oil, and sulfuric acid. Fungicides mainly controlled the genera Alternaria, Aspergillus, Bipolaris, Chaetomium, Cladosporium, Colletotrichum, Curvularia, Fusarium, Penicillium, Phoma and Rhizopus. Essential oils showed positive effects in controlling Bipolaris, Curvularia, and Fusarium. Sulfuric acid reduced the incidence of Bipolaris, Colletotrichum, Curvularia, Fusarium, Phoma, and Rhizopus. Seed treatment is one of the promising strategies for obtaining seeds of a healthy quality. As a result, the fungicides and botanical components listed above were promising in reducing fungi associated with forage seeds and can be used for seed treatments and in the development of new products with a natural base through essential oils.

Improved node culture methods for rapid vegetative propagation of switchgrass (Panicum virgatum L.).

BACKGROUND: Switchgrass (Panicum virgatum L.) is an important bioenergy and forage crop. The outcrossing nature of switchgrass makes it infeasible to maintain a genotype through sexual propagation. Current asexual propagation protocols in switchgrass have various limitations. An easy and highly-efficient vegetative propagation method is needed to propagate large natural collections of switchgrass genotypes for genome-wide association studies (GWAS). RESULTS: Micropropagation by node culture was found to be a rapid method for vegetative propagation of switchgrass. Bacterial and fungal contamination during node culture is a major cause for cultural failure. Adding the biocide, Plant Preservative Mixture (PPM, 0.2%), and the fungicide, Benomyl (5 mg/l), in the incubation solution after surface sterilization and in the culture medium significantly decreased bacterial and fungal contamination. In addition, "shoot trimming" before subculture had a positive effect on shoot multiplication for most genotypes tested. Using the optimized node culture procedure, we successfully propagated 330 genotypes from a switchgrass GWAS panel in three separate experiments. Large variations in shoot induction efficiency and shoot growth were observed among genotypes. Separately, we developed an in planta node culture method by stimulating the growth of aerial axillary buds into shoots directly on the parent plants, through which rooted plants can be generated within 6 weeks. By circumventing the tissue culture step and avoiding application of exterior hormones, the in planta node culture method is labor- and cost-efficient, easy to master, and has a high success rate. Plants generated by the in planta node culture method are similar to seedlings and can be used directly for various experiments. CONCLUSIONS: In this study, we optimized a switchgrass node culture protocol by minimizing bacterial and fungal contamination and increasing shoot multiplication. With this improved protocol, we successfully propagated three quarters of the genotypes in a diverse switchgrass GWAS panel. Furthermore, we established a novel and high-throughput in planta node culture method. Together, these methods provide better options for researchers to accelerate vegetative propagation of switchgrass.

Sulfur supply reduces barium toxicity in Tanzania guinea grass (Panicum maximum) by inducing antioxidant enzymes and proline metabolism.

Sulfur (S) can play essential roles in protecting plants against abiotic stress, including heavy metal toxicity. However, the effect of this nutrient on plants exposed to barium (Ba) is still unknown. This study was designed to evaluate the S supply on oxidative stress and the antioxidant system of Tanzania guinea grass under exposure to Ba, grown in a nutrient solution under greenhouse conditions. It was studied the influence of S/Ba combinations in nutrient solution on oxidative stress indicators (hydrogen peroxide, malondialdehyde, and proline) and antioxidant enzyme activities (superoxide dismutase, catalase, ascorbate peroxidase, guaiacol peroxidase, and glutathione reductase). The treatments consisted in thirteen S/Ba combinations in the nutrient solution (0.1/0.0; 0.1/5.0; 0.1/20.0; 1.0/2.5; 1.0/10.0; 1.9/0.0 - control; 1.9/5.0; 1.9/20.0; 2.8/2.5; 2.8/10.0; 3.7/0.0; 3.7/5.0 and 3.7/20.0 mM of S and Ba, respectively). The plants were grown for two growth periods, which consisted of fourteen days of S supply and the eight days of Ba exposure each one. The severe S deficiency decreased the superoxide dismutase activity, regardless of Ba exposure in recently expanded leaves and culms plus sheaths. However, supplemental S supply (above 1.9 mM S, which corresponds to S supply adequate to plant growth) it improved the superoxide dismutase activity in these tissues under high Ba concentrations. Conversely, the severe S deficiency increased the activities of catalase, ascorbate peroxidase, and glutathione reductase in grass leaves slightly, without Ba exposure influence. It was observed that the supplemental S supply also induced the guaiacol peroxidase activity and proline production in culms plus sheaths under high Ba rates, showing values until 2.5 and 3.1 folds higher than the control treatment, respectively. In plants under exposure to 20.0 mM Ba, the supplemental S supply decreased the malondialdehyde content in culms plus sheaths in 17% compared to 1.9 mM S. These results indicate that supplemental S supply can mitigate Ba toxicity in Tanzania guinea grass, mainly by improving superoxide dismutase and guaiacol peroxidase activities, and proline metabolism.

Sulfamethoxazole sorption by cattail and switchgrass roots.

Sorption to roots is one of several mechanisms by which plant-assisted attenuation of antibiotics can be achieved. The objectives of this study were to (1) evaluate the sorption of sulfamethoxazole (SMX) by cattail and switchgrass roots, (2) determine the kinetics of SMX sorption by cattail and switchgrass roots, and (3) characterize the temperature-dependency of SMX sorption. A batch sorption experiment was conducted to measure SMX sorption by roots of the two plant species using five initial antibiotic concentrations (2.5, 5, 10, 15, and 20 µg L-1) and eight sampling times (0, 0.5, 1, 2, 4, 8, 12, and 24 h). Another batch experiment was conducted at three temperatures (5, 15, and 25 °C) to determine the effect of temperature on sorption kinetics. SMX sorption followed pseudo-second-order kinetics. The pseudo-second-order rate constant (k2) decreased with increasing temperature for both plant species. The rate constant followed the order: 5 °C = 15 °C > 25 °C for cattail and 5 °C > 15 °C = 25 °C for switchgrass. Results from this study show that switchgrass roots are more effective than cattail roots in the removal of SMX. Therefore, the use of switchgrass in systems designed for phytoremediation of contaminants might also provide an efficient removal of some antibiotics.

Green fabricated zinc oxide nanoformulated media enhanced callus induction and regeneration dynamics of Panicum virgatum L.

The current study focuses on the usage of bio synthesized zinc oxide nanoparticles to increase the tissue culture efficiency of important forage grass Panicum virgatum. Zinc being a micronutrient enhanced the callogenesis and regeneration efficiency of Panicum virgatum at different concentrations. Here, we synthesized zinc oxide nanoparticles through Cymbopogon citratus leaves extract to evaluate the effect of zinc oxide nanoparticles on plant regeneration ability in switchgrass. X-ray diffraction (XRD) and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) validate phase purity of green synthesize Zinc oxide nanoparticles whereas, electron microscopy (SEM) has illustrated the average size of particle 50±4 nm with hexagonal rod like shape. Energy dispersive spectroscopy X-ray (EDS) depicted major peaks of Zn (92.68%) while minor peaks refer to Oxygen (7.32%). ZnO-NPs demonstrated the incredibly promising results against callogenesis. Biosynthesized ZnO-NPs at optimum concentration showed very promising effect on plant regeneration ability. Both the explants, seeds and nodes showed dose dependent response and upon high doses exceeding 40 mg/L the results were recorded negative, whereas at 30 mg/L both explants demonstrated 70% and 76% regeneration frequency. The results conclude that ZnO-NPs enhance the plant growth and development and tailored the nutritive properties at nano-scale. Furthermore, eco-friendly approach of ZnO-NPs synthesis is strongly believed to improve in vitro regeneration frequencies in several other monocot plants.

Identification and Validation of Reference Genes for RT-qPCR Analysis in Switchgrass under Heavy Metal Stresses.

Switchgrass (Panicum Virgatum L.) has been recognized as the new energy plant, which makes it ideal for the development of phytoremediation on heavy metal contamination in soils with great potential. This study aimed to screen the best internal reference genes for the real-time quantitative PCR (RT-qPCR) in leaves and roots of switchgrass for investigating its response to various heavy metals, such as cadmium (Cd), lead (Pb), mercury (Hg), chromium (Cr), and arsenic (As). The stability of fourteen candidate reference genes was evaluated by BestKeeper, GeNorm, NormFinder, and RefFinder software. Our results identified U2AF as the best reference gene in Cd, Hg, Cr, and As treated leaves as well as in Hg, Pb, As, and Cr stressed root tissues. In Pb treated leaf tissues, 18S rRNA was demonstrated to be the best reference gene. CYP5 was determined to be the optimal reference gene in Cd treated root tissues. The least stable reference gene was identified to be CYP2 in all tested samples except for root tissues stressed by Pb. To further validate the initial screening results, we used the different sets of combinatory internal reference genes to analyze the expression of two metal transport associated genes (PvZIP4 and PvPDB8) in young leaves and roots of switchgrass. Our results demonstrated that the relative expression of the target genes consistently changed during the treatment when CYP5/UBQ1, U2AF/ACT12, eEF1a/U2AF, or 18S rRNA/ACT12 were combined as the internal reference genes. However, the time-dependent change pattern of the target genes was significantly altered when CYP2 was used as the internal reference gene. Therefore, the selection of the internal reference genes appropriate for specific experimental conditions is critical to ensure the accuracy and reliability of RT-qPCR. Our findings established a solid foundation to further study the gene regulatory network of switchgrass in response to heavy metal stress.

Elevated CO2 and warming change the nutrient status and use efficiency of Panicum maximum Jacq.

Panicum maximum Jacq. 'Mombaça' (Guinea grass) is a C4 forage grass widely used in tropical pastures for cattle feeding. In this study, we evaluated the isolated and combined effects of warming and elevated CO2 concentration [CO2] during summer on nutrient content, nutrient accumulation, nutrient use efficiency and growth of P. maximum under field conditions. Field temperature and [CO2] were controlled by temperature free-air controlled enhancement and free-air CO2 enrichment systems, respectively. We tested two levels of canopy temperature: ambient temperature (aT) and 2°C above ambient temperature (eT), as well as two levels of atmospheric [CO2]: ambient [CO2] (aCO2) and 200 ppm above ambient CO2 (eCO2). The experiment was established in a completely randomized design with four replications, in a 2×2 factorial scheme. After pasture establishment, plants were exposed to the treatments during 30 days, with evaluations at 9, 16, 23 and 30 days after the treatments started. Results were dependent on the time of the evaluation, but in the last evaluation (beginning of the grazing), contents of N, K, Mg and S did not change as a function of treatments. However, P decreased as a function of warming under both levels of [CO2], and Ca increased under [eCO2] combined with warming. There was an increase in root dry mass under warming treatment. Combined treatment increased N, Ca and S accumulation without a corresponding increase in the use efficiency of these same nutrients, indicating that the fertiliser dose should increase in the next decades due to climate change. Our short-term results in young and well fertilized pasture suggest that under the combination of [eCO2] and eT conditions, P. maximum productivity will increase and the nutritional requirement for N, Ca and S will also increase.

Potassium affects the phytoextraction potential of Tanzania guinea grass under cadmium stress.

The supply of potassium (K) is a strategy to increase the tolerance of plants exposed to Cd toxicity. The aim of this study was to verify the influence of K on the growth and potential of Tanzania guinea grass (Panicum maximum Jacq. cv. Tanzania (syn. Megathyrsus maximus (Jacq.) B.K. Simon & S.W.L. Jacobs)) for Cd phytoextraction as well as to evaluate nutritional attributes of this grass under conditions of Cd stress. The experiment was conducted in a randomized complete block design, using a 3 × 4 factorial arrangement, with three replications. Three rates of K (0.4, 6.0, and 11.6 mmol L-1) were combined with four rates of Cd (0.0, 0.5, 1.0, and 1.5 mmol L-1) in nutrient solution. Two plant growth periods were evaluated. The increase in K supply to plants exposed to Cd rates of up to 1.0 mmol L-1 caused increase in morphogenic and production attributes, as well as reduction in tiller mortality rate, in the second growth period. K concentrations (in both harvests) increased, while calcium and magnesium concentrations in the second harvest decreased with increasing Cd rates. The high availability of Cd (1.5 mmol L-1) in the nutrient solution caused decrease in relative chlorophyll index (RCI) in both harvests. The high supply of K to plants exposed to Cd resulted in high shoot dry mass production, reducing Cd concentration in the photosynthetic tissues (which means great tolerance of the plant) and increasing the accumulation of this metal in the shoots that can be harvested. Therefore, K increases the Cd phytoextraction capacity of Tanzania guinea grass.

Ozone phytotoxicity to Panicum maximum and Cenchrus ciliaris at Indo-Gangetic plains: an assessment of antioxidative defense and growth responses.

Two common tropical grassland species, Panicum maximum Jacq. (Guinea grass) and Cenchrus ciliaris (Buffel grass) of Indo-Gangetic plains were assessed for their responses under future level of O3 (ambient +30 ppb) using open top chambers. Plants were assessed for foliar injuries, pigments, growth, biomass accumulation, histochemical localization of reactive oxygen species (ROS), antioxidant defense system and ROS scavenging activities at two stages. Foliar injuries were noticed at an early stage in P. maximum compared to C. ciliaris. Significant reductions were observed in total chlorophyll, growth and total biomass in both species. Significant increases in contents of melondialdehyde and ascorbic acid in P. maximum while total phenolics and thiols in C. ciliaris were found. Histochemical analysis showed more production of superoxide radicals and hydrogen peroxide in leaf tissues of P. maximum compared to C. ciliaris. It can be concluded that higher level of primary antioxidants (total phenolics and thiols) along with superoxide dismutase and ascorbate peroxidase scavenged O3 effectively in C. ciliaris causing less reduction of biomass which is used as a feed for cattles. In P. maximum, more photosynthates were allocated for defense, leading to higher reduction in total biomass compared to C. ciliaris. The leaf area ratio was higher in P. maximum compared to C. ciliaris under elevated O3. The study further suggests higher susceptibility of P. maximum compared to C. ciliaris under future level of O3 exposure.

Partial replacement of nitrate by ammonium increases photosynthesis and reduces oxidative stress in tanzania guinea grass exposed to cadmium.

In order to grow and effectively uptake and accumulate cadmium (Cd), plants used for phytoextraction have to cope with toxicity, which may be influenced by the supply of nitrate (NO3-) and ammonium (NH4+). Thus, we evaluated the effect of these nitrogen forms on the photosynthetic and antioxidant enzyme activities of Panicum maximum cv. Tanzania (tanzania guinea grass) under Cd stress. Plants were grown in nutrient solution under greenhouse conditions and subjected to a 3 × 3 factorial experiment. They were supplied with three NO3-/NH4+ ratios (100/0, 70/30 and 50/50) and exposed to three Cd rates (0.0, 0.5 and 1.0 mmol L-1), being arranged in a randomized complete block design with three replications. Gas exchange parameters, oxidative stress indicators, proline concentration and antioxidant enzyme activities were studied. Exposure to Cd reduced photosynthesis by causing stomatal closure and impairing electron transport. However, the simultaneous supply of NO3- and NH4+, particularly at a 50/50 ratio, restored gas exchange and improved the function of photosystem II, increasing the photosynthetic capacity of the grass. Plants grown with 50/50 showed reduced lipid peroxidation along with increased proline synthesis. Moreover, this NO3-/NH4+ ratio increased the tolerance of tanzania guinea grass to Cd by inducing high superoxide dismutase and glutathione reductase activities in shoots and roots, respectively, maintaining cellular homeostasis and reducing oxidative stress. The negative effects of Cd on photosynthesis and on the balance between oxidants and antioxidants are attenuated by the partial replacement of NO3- by NH4+ in the nutrient solution.

Effects of water availability and UV radiation on silicon accumulation in the C4 crop proso millet.

Proso millet (Panicum miliaceum L.) is an annual thermophilic, drought-resistant, short-season C4 grass that is suitable for growing under the present changing meteorological conditions. However, water shortage can reduce plant production and hinder mineral nutrition in plants, including silicon, which is of crucial importance for grasses. Ultraviolet radiation can mitigate the impacts of water shortage, and therefore we examined the effects of moderate water shortage and reduced ultraviolet radiation on different leaf traits, including leaf levels of silicon and other elements, and plant biomass production. Moderate water shortage and ambient ultraviolet radiation did not affect the contents of photosynthetic pigments, while they reduced stomata density. Water shortage significantly decreased leaf light reflectance in the ultraviolet and violet regions. Leaf silicon, calcium, phosphorus, and sulphur levels were significantly lower with reduced water availability and significantly higher with reduced ultraviolet radiation. Leaf silicon levels ranged from 1.5% to 2.5% of leaf dry mass, with leaf calcium levels from 0.3% to 0.6%. Except for chlorine and potassium, the levels of these elements in the leaves were significantly positively related (p ≤ 0.05) to soil moisture levels. Water availability, but not ultraviolet radiation, significantly reduced living leaf biomass.

Nitrate and ammonium proportion plays a key role in copper phytoextraction, improving the antioxidant defense in Tanzania guinea grass.

Various nitrate and ammonium proportions (NO3-/NH4+) in the growth media can increase metal phytoextraction compared to supplying solely NO3-. However, there are no studies showing these effects in plants under copper (Cu) contamination as well as their consequences in plant stress tolerance. The objective was to evaluate the effect of NO3-/NH4+ proportions in Cu phytoextraction by Panicum maximum cv. Tanzania and its consequence in the oxidative stress, photosynthesis, and antioxidant system under Cu stress. The experiment was carried out in a randomized complete block design, by using a 3 × 4 factorial with six replications. Three NO3-/NH4+ proportions (100/0, 70/30, and 50/50) were combined with four Cu rates (0.3, 250, 500, and 1000 µmol L-1) in the nutrient solution. It was found that the largest Cu accumulation in the shoots occurred at the first harvest of the plants supplied with 70/30 NO3-/NH4+ and Cu 1000 µmol L-1. Such plants also displayed high concentrations of proline in the shoots as well as high superoxide dismutase activity in the roots. Malondialdehyde concentration was high in the plant parts at the Cu rate of 1000 µmol L-1. Hence, transpiration rates, stomatal conductance, quantum efficiency of photosystem II, electron transport rate, and net photosynthesis were all low at the Cu rate of 1000 µmol L-1. Catalase, guaiacol peroxidase, ascorbate peroxidase, and glutathione reductase activities in the roots were high when plants were exposed to Cu 1000 µmol L-1. In conclusion, the combination of NO3- with NH4+ increases copper phytoextraction that causes oxidative stress, but also favors the antioxidant system of Tanzania guinea grass in attempt to tolerate such stress.

Nanoparticle titanium dioxide affects the growth and microRNA expression of switchgrass (Panicum virgatum).

Nanoparticle TiO2 is a common chemical used in daily life. As increasing usage of TiO2, it is becoming a potentially dangerous contaminant to the environment. However, the impact of TiO2 is not well understood. In this paper, switchgrass was employed to investigate the impacts of nanoparticle TiO2 on plant growth and development as well as the potential impact on the expression of microRNAs (miRNAs). TiO2 significantly affected switchgrass seed generation as well as plant growth and development in a dose-dependent manner. Particularly, TiO2 significantly inhibited root development. miRNA expressions were also significantly altered. Nanoparticle TiO2 may regulate plant development through controlling the expression of certain miRNAs. Among the 16 tested miRNAs, the expression of some miRNAs, such as miR390 and miR399 was increased with increasing TiO2 concentrations; the expression of some miRNAs, such as miR169 was decreased with increasing TiO2 concentrations; the other miRNAs show different expression patterns.

Effects of carbon-based nanomaterials on seed germination, biomass accumulation and salt stress response of bioenergy crops.

Bioenergy crops are an attractive option for use in energy production. A good plant candidate for bioenergy applications should produce a high amount of biomass and resist harsh environmental conditions. Carbon-based nanomaterials (CBNs) have been described as promising seed germination and plant growth regulators. In this paper, we tested the impact of two CBNs: graphene and multi-walled carbon nanotubes (CNTs) on germination and biomass production of two major bioenergy crops (sorghum and switchgrass). The application of graphene and CNTs increased the germination rate of switchgrass seeds and led to an early germination of sorghum seeds. The exposure of switchgrass to graphene (200 mg/l) resulted in a 28% increase of total biomass produced compared to untreated plants. We tested the impact of CBNs on bioenergy crops under salt stress conditions and discovered that CBNs can significantly reduce symptoms of salt stress imposed by the addition of NaCl into the growth medium. Using an ion selective electrode, we demonstrated that the concentration of Na+ ions in NaCl solution can be significantly decreased by the addition of CNTs to the salt solution. Our data confirmed the potential of CBNs as plant growth regulators for non-food crops and demonstrated the role of CBNs in the protection of plants against salt stress by desalination of saline growth medium.

Transcriptome analysis of Cd-treated switchgrass root revealed novel transcripts and the importance of HSF/HSP network in switchgrass Cd tolerance.

KEY MESSAGE: Transcriptome analysis of Cd-treated switchgrass roots not only revealed novel switchgrass transcripts and gene structures but also highlighted the indispensable role of HSF/HSP network in switchgrass Cd tolerance. Switchgrass (Panicum virgatum L.), a C4 perennial tall grass, can be used for revegetation of Cd-contaminated soil. In the present study, a comparative transcriptome analysis of Cd-treated switchgrass roots was conducted. The result revealed a total of 462 novel transcripts and refined gene structures of 2337 transcripts. KEGG pathway and Gene Ontology analyses of the differentially expressed genes (DEGs) suggested that activation of redox homeostasis and oxidation-related metabolic processes were the primary response to Cd stress in switchgrass roots. In particular, 21 out of 23 differentially expressed shock transcription factor genes (HSFs), and 22 out of 23 differentially expressed heat shock protein genes (HSPs) had increased expression levels after Cd treatment. Furthermore, over-expressing one HSP-encoding gene in Arabidopsis significantly improved plant Cd tolerance. The result highlighted the activation of the redox homeostasis and the involvement of the HSF/HSP network in re-establishing normal protein conformation and thus cellular homeostasis in switchgrass upon Cd stress. These DEGs, especially those of the HSF/HSP network, could be used as candidate genes for further functional studies toward improved plant Cd tolerance in switchgrass and related species.

Overexpression of gene encoding the key enzyme involved in proline-biosynthesis (PuP5CS) to improve salt tolerance in switchgrass (Panicum virgatum L.).

KEY MESSAGE: Genetic improvement through overexpressing PuP5CS in switchgrass is feasible for enhancing plant salt stress tolerance. Switchgrass (Panicum virgatum L.) has developed into a dedicated bioenergy crop. To improve the biomass production of switchgrass grown on different types of soil, abiotic stress tolerance traits are considered for its genetic improvement. Proline accumulation is a widespread response when plants are subjected to abiotic stresses such as drought, cold and salinity. In plants, P5CS gene encodes the key regulatory enzyme that plays a crucial role in proline biosynthesis. Here, we introduced the PuP5CS gene (from Puccinellia chinampoensis) into switchgrass by Agrobacterium-mediated transformation. Transgenic lines overexpressing the PuP5CS gene showed phenotypic advantages, in leaf width, internode diameter, internode length, tiller numbers and precocious flowering under normal conditions, and the transgenic lines displayed better regenerative capacity in forming more tillers after harvest. Moreover, the PuP5CS gene enhanced the salt tolerance of transgenic switchgrass by altering a wide range of physiological responses. In accordance with the physiological results, histological analysis of cross sections through the leaf blade showed that the areas of bulliform cells and bundle sheath cells were significantly increased in PuP5CS-overexpressing leaves. The expression levels of ROS scavenging-associated genes in transgenic plants were higher than in control plants under salt stress. The results show that genetic improvement through overexpressing PuP5CS in switchgrass is feasible for enhancing plant stress tolerance.

Transcriptome-based analyses of phosphite-mediated suppression of rust pathogens Puccinia emaculata and Phakopsora pachyrhizi and functional characterization of selected fungal target genes.

Phosphite (Phi) is used commercially to manage diseases mainly caused by oomycetes, primarily due to its low cost compared with other fungicides and its persistent control of oomycetous pathogens. We explored the use of Phi in controlling the fungal pathogens Puccinia emaculata and Phakopsora pachyrhizi, the causal agents of switchgrass rust and Asian soybean rust, respectively. Phi primes host defenses and efficiently inhibits the growth of P. emaculata, P. pachyrhizi and several other fungal pathogens tested. To understand these Phi-mediated effects, a detailed molecular analysis was undertaken in both the host and the pathogen. Transcriptomic studies in switchgrass revealed that Phi activates plant defense signaling as early as 1 h after application by increasing the expression of several cytoplasmic and membrane receptor-like kinases and defense-related genes within 24 h of application. Unlike in oomycetes, RNA sequencing of P. emaculata and P. pachyrhizi did not exhibit Phi-mediated retardation of cell wall biosynthesis. The genes with reduced expression in either or both rust fungi belonged to functional categories such as ribosomal protein, actin, RNA-dependent RNA polymerase, and aldehyde dehydrogenase. A few P. emaculata genes that had reduced expression upon Phi treatment were further characterized. Application of double-stranded RNAs specific to P. emaculata genes encoding glutamate N-acetyltransferase and cystathionine gamma-synthase to switchgrass leaves resulted in reduced disease severity upon P. emaculata inoculation, suggesting their role in pathogen survival and/or pathogenesis.

Enhanced Cold Tolerance and Tillering in Switchgrass (Panicum virgatum L.) by Heterologous Expression of Osa-miR393a.

The microRNA393 (miR393) family is one of the conserved miRNA families in the plant kingdom. MiR393 was reported to regulate rice tillering and abiotic stress resistance positively through an auxin signaling pathway. However, little is known about the function of miR393 in switchgrass (Panicum virgatum L.), an important bioenergy C4 grass plant. We tested the expression level of miR393 and its four putative target genes (PvAFB1, PvAFB2, PvAFB3 and PvTIR1) in switchgrass, and found that these genes all responded to cold stress and exogenous 1-naphthaleneacetic acid (NAA) treatment. To investigate the function of miR393 in switchgrass, we enhanced miR393 expression by introducing an Osa-miR393a gene into switchgrass. The results showed that cold tolerance of the transgenic T0 and T1 generation plants was highly improved. Cold tolerance-related genes PvCOR47, PvICE1 and PvRAV1 were negatively regulated by exogenous NAA, and the expression of these genes was significantly higher in transgenic plants than in wild-type plants. The transgenic T1 seedlings were more tolerant to exogenous NAA treatment, accumulating less H2O2 after cold treatments. It was also observed that the miR393/target module regulates cold tolerance responses in Arabidopsis. In addition, transgenic plants overexpressing miR393 had significantly more tillers and higher biomass yield per plant in greenhouse and field tests. Forage quality analyses revealed that the soluble sugar contents of transgenic plants were increased markedly. Overall, the results suggested that overexpression of miR393 improved cold tolerance and tillering of switchgrass through regulation of auxin signaling transduction.